Vaccines for treatment of lymphoma and leukemia

ABSTRACT

The present invention provides multivalent vaccines for the treatment of B-cell malignancies (e.g., lymphomas and leukemias). The present invention also provides methods for the production of custom vaccines, including multivalent vaccines for the treatment of immune cell tumors malignancies as well as methods of treating immune cell tumors using custom vaccines.

This is a Continuation-In-Part of application Ser. No. 08/644,664 filedMay 1, 1996 (now U.S. Pat. No. 5,776,746).

FIELD OF THE INVENTION

The present invention generally relates to improved methods for theamplification and expression of recombinant genes in cells. Theamplified cells provide large quantities of recombinant proteinssuitable for immunotherapy for treatment of lymphomas and leukemias.

BACKGROUND OF THE INVENTION

As an increasing number of genes are isolated and developed for theexpression of a wide array of useful polypeptide drugs, there is anincreasing need to enhance the efficiencies and economies of theprocess. It is advantageous to obtain such polypeptides from mammaliancells since such polypeptides or proteins are generally correctlyfolded, appropriately modified and completely functional, often inmarked contrast to those proteins as expressed in bacterial cells.

When large amounts of product are required, it is necessary to identifycell clones in which the vector sequences are maintained (i.e.,retained) during cell proliferation. Such stable vector maintenance canbe achieved either as a consequence of integration of the vector intothe DNA of the host cell or by use of a viral replicon such as bovinepapillomavirus (BPV).

The use of viral vectors such as BPV-based vectors for the generation ofstable cell lines expressing large amounts of a recombinant protein hasbeen successful in some cases; however, the use of viral vectors islimited by the fact that the viral vectors are restricted in the celltypes in which they can replicate. Furthermore expression levels andepisomal maintenance of the viral vector can be influenced by the DNAsequences inserted into the vector.

Where the vector has been integrated into the genomic DNA of the hostcell to improve stability, the copy number of the vector DNA, andconcomitantly the amount of product which could be expressed, can beincreased by selecting for cell lines in which the vector sequences havebeen amplified after integration into the DNA of the host cell.

A known method for carrying out such a selection procedure is totransform a host cell with a vector comprising a DNA sequence whichencodes an enzyme which is inhibited by a known drug. The vector mayalso comprise a DNA sequence which encodes a desired protein.Alternatively the host cell may be co-transformed with a second vectorwhich comprises the DNA sequence which encodes the desired protein.

The transformed or co-transformed host cells are then cultured inincreasing concentrations of the known drug hereby selectingdrug-resistant cells. It has been found that one common mechanismleading to the appearance of mutant cells which can survive in theincreased concentrations of the otherwise toxic drug is theover-production of the enzyme which is inhibited by the drug. This mostcommonly results from increased levels of its particular MRNA, which inturn is frequently caused by amplification of vector DNA and hence genecopies.

It has also been found that when drug resistance is caused by anincrease in copy number of the vector DNA encoding the inhibitableenzyme, there is a concomitant increase in the copy number of the vectorDNA encoding the desired protein in the DNA of the host cell. There isthus an increased level of production of the desired protein.

The most commonly used system for such co-amplification usesdihydrofolate reductase (DHFR) as the inhibitable enzyme. This enzymecan be inhibited by the drug methotrexate (MTX). To achieveco-amplification, a host cell which lacks an active gene which encodesDHFR is either transformed with a vector which comprises DNA sequencesencoding DHFR and a desired protein or co-transformed with a vectorcomprising a DNA sequence encoding DHFR and a vector comprising a DNAsequence encoding the desired protein. The transformed or co-transformedhost cells are cultured in media containing increasing levels of MTX,and those cell lines which survive are selected.

The co-amplification systems which are presently available suffer from anumber of disadvantages. For instance, it is generally necessary to usea host cell which lacks an active gene encoding the enzyme which can beinhibited. This tends to limit the number of cell lines which can beused with any particular co-amplification system.

For instance, there are at present, only two cell lines known which lackthe gene encoding DHFR and both of these cell lines are derivatives ofthe CHO-K1 cell line. These DHFR⁻ CHO cell lines cannot be used toexpress certain protein products at high levels because CHO cells lackspecialized postranslational modification pathways. For example, theproduction of functional human protein C requires that the cell possessthe vitamin K-dependent y-carboxylation pathway; CHO cells cannotproperly modify the human protein C protein [Walls et al., (1989) Gene81:139].

Attempts to use DHFR genes as dominant selectable markers in other celllines (i.e., cell lines synthesizing wild type levels of DHFR) has notproved satisfactory. For instance, a MTX-resistant mutant DHFR or a DHFRgene under the control of a very strong promoter can act as a dominantselectable marker in certain cell types but such high concentrations ofMTX are required that it has not been possible to achieve high copynumbers by selection for gene amplification using current methodologies.

Another approach to allow the use of DHFR as a dominant selectablemarker in DHFR⁺ cell lines is the use of both the DHFR gene and a geneencoding a selectable marker, such as the hygromycin phosphotransferase(hyg) gene, in addition to the gene of interest [Walls, et al. (1989),supra]. This approach is used to circumvent the problem of amplificationof the endogenous dhfr gene during selection with MTX. The cells aretransfected with DNA encoding the three genes and the cells are firstselected for their ability to grow in hygromycin. The cells are thenselected for the ability to grow in increasing concentrations of MTX.While this approach allows for the co-amplification of genes in dhfr⁺cell lines, present protocols show that the dhfr gene is amplified to ahigher degree than the gene of interest with successive rounds ofamplification (i.e., stepwise increases in MTX concentration). Forexample, in several amplified clones the dhfr gene was present atapproximately 100 copies while the gene of interest was present at only20 copies.

Clearly, the art needs improved methods which would consistently providefor the coincidental amplification of the amplifiable marker and thegene of interest in a variety of cell lines. Furthermore, the art needsa means of amplfying DNA sequences of interest which is efficient,reproducible and which is not limited to the use of specialized enzymedeficient host cell lines or to a limited number of cell lines.

Improved methods which consistently provide for the coincidentalamplification of the amplifiable marker and the gene of interest in avariety of cell lines and which are efficient and reproducible wouldallow the production of custom tumor-specific vaccines on a scalecommensurate with patient demand. Current methods for producing customtumor vaccines for the treatment of B-cell lymphoma are insufficient tomeet current and anticipated future demand.

SUMMARY OF THE INVENTION

The present invention provides methods for the production of cell linescontaining amplified copies of recombinant DNA sequences. Because theamplified cell lines contain several different recombinant DNA sequences(e.g., the amplification vector, one or more expression vectors andoptionally a selection vector) which are coordinately amplified, thecell lines are said to have co-amplified the input or exogenous DNAsequences. The methods of the present invention permit the efficientisolation of the desired amplified cell lines with a considerablesavings in time relative to existing amplification protocols. The geneamplification methods of the present invention permit the production ofcustom vaccines, including multivalent vaccines, which are useful forthe treatment of immune cell tumors (e.g., lymphomas and leukemias).

In one embodiment, the present invention provides a multivalent vaccinecomprising at least two recombinant variable regions of immunoglobulinmolecules derived from B-cell lymphoma cells, wherein said cells expressat least two different immunoglobulin molecules, said immunoglobulinmolecules differing by at least one idiotope. The invention is notlimited by the context in which the recombinant variable regions areutilized; the variable regions may be present within an entirerecombinant immunoglobulin (Ig) molecule, they may be present on Fab,Fab' or F(ab')₂ fragments (which may be generated by cleavage of therecombinant Ig molecule or they may be produced using molecularbiological means) or they may be present on single chain antibody (Fv)molecules. In a preferred embodiment, the multivalent vaccine comprisesat least two recombinant immunoglobulin molecules comprising saidrecombinant variable regions derived from said lymphoma cells.

In one embodiment, the immunoglobulin molecules comprising recombinantvariable regions derived from a patient's lymphoma cells are covalentlylinked to an immune-enhancing cytokine. The linkage of the cytokine tothe Ig molecule may be achieved by a variety of means known to the artincluding conventional coupling techniques (e.g., coupling withdehydrating agents such as dicyclohexylcarbodiimide (DCCI), ECDI and thelike), the use of linkers capable of coupling through sulfhydryl groups,amino groups or carboxyl groups (available from Pierce Chemical Co.,Rockford, Ill.), by reductive amination. In addition, the covalentlinkage may be achieved by molecular biological means (e.g., theproduction of a fusion protein using an expression vector comprising anucleotide sequence encoding the recombinant Ig operably linked to anucleotide sequence encoding the desired cytokine).

The invention is not limited by the immune-enhancing cytokine employed.In a preferred embodiment, the cytokine is selected from the groupconsisting of granulocyte-macrophage colony stimulating factor,interleukin-2 and interleukin-4.

In one embodiment, the multivalent vaccines of the present inventioncomprise at least one pharmaceutically acceptable excipient. Theinvention is not limited by the nature of the excipient employed. Thepharmaceutical compositions of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks's solution, Ringer's solution, or physiologically bufferedsaline. Aqueous injection suspensions may contain substances whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Additionally, suspensions of the activecompounds may be prepared as appropriate oily injection suspensions.Suitable lipophilic solvents or vehicles include fatty oils such assesame oil, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides, or liposomes. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

In a preferred embodiment, the multivalent vaccine further comprises anadjuvant. When the vaccine is to be administered to a human subject,adjuvants approved for use in humans are employed (e.g., SAF-1, alum,etc.). The recombinant Ig proteins (including fragments of Ig proteins)which comprise the multivalent vaccine may be conjugated to a carrierprotein such as KLH.

The present invention also provides a method of producing a vaccine fortreatment of B-cell lymphoma comprising: a) providing: i) malignantcells isolated from a patient having a B-cell lymphoma; ii) anamplification vector comprising a recombinant oligonucleotide having asequence encoding a first inhibitable enzyme operably linked to aheterologous promoter; iii) a eukaryotic parent cell line; b) isolatingfrom the malignant cells nucleotide sequences encoding at least oneV_(H) region and at least one V_(L) region, the V_(H) and V_(L) regionsderived from immunoglobulin molecules expressed by the malignant cells;c) inserting the nucleotide sequences encoding the V_(H) and V_(L)regions into at least one expression vector; d) introducing theexpression vector(s) and the amplification vector into the parent cellto generate one or more transformed cells; e) growing the transformedcell(s) in a first aqueous solution containing an inhibitor capable ofinhibiting the inhibitable enzyme wherein the concentration of theinhibitor present in the first aqueous solution is sufficient to preventgrowth of the parent cell line; and f) identifying a transformed cellcapable of growth in the first aqueous solution, wherein the transformedcell(s) capable of growth expresses the V_(H) and V_(L) regions. In apreferred embodiment, the transformed cell capable of growth in thefirst aqueous solution contains an amplified number of copies of theexpression vector(s) and an amplified number of copies of theamplification vector.

In another preferred embodiment, the nucleotide sequences encoding theV_(H) and C_(L) regions comprise at least two V_(H) and at least twoC_(L) regions (in this manner, a multivalent vaccine is produced).

The method of the present invention is not limited by the nature of themeans employed to introduce the vectors into the parent cell line. Theart is well aware of numerous methods which allow the introduction ofexogenous DNA sequences into mammalian cells, including but not limitedto electroporation, microinjection, lipofection, protoplast fusion,liposome fusion and the like. In a preferred embodiment, the vectors areintroduced into the parent cell line by electroporation.

The present invention is not limited by the nature of the cell linechosen as the parent cell line; a variety of mammalian cell lines may beemployed including CHO cell lines and variants thereof, mouse L cellsand BW5147 cells and variants thereof. The chosen cell line grow ineither an attachment-dependent or attachment-independent manner. In apreferred embodiment, the parent cell line is a T lymphoid cell line; aparticularly preferred T lymphoid cell line is the BW5147.G.1.4 cellline.

In another embodiment, the method of the present invention employs aparent cell line which contains an endogenous gene encoding a secondinhibitable enzyme (e.g., the genome of the parent cell line contains anendogenous gene comprising a coding region encoding a second inhibitableenzyme which is operably linked to the promoter naturally linked to thiscoding region (i.e., the endogenous promoter for this gene). A contrastis made between the input or exogenous recombinant sequences encodingthe first inhibitable enzyme and an endogenous gene encoding aninhibitable enzyme. The endogenous gene sequences will be expressedunder the control of the endogenous promoter. Typically, theamplification vector will comprise a sequence encoding an inhibitableenzyme operably linked to a heterologous (i.e., not the endogenous)promoter. The sequences encoding the first and the second inhibitableenzyme may encode the same or a different enzyme. Furthermore, when thesame enzyme is encoded by the two sequences (ie., the recombinant andthe endogenous sequences), these sequences may be derived from the sameor a different source (ie., the recombinant sequence may encode anenzyme isolated from a mouse cell and may introduced into a mouse cellline which contains an endogenous gene encoding the same enzyme;alternatively, the recombinant sequence may encode an enzyme derivedfrom a different species than that of the parent cell line (e.g., therecombinant sequence may encode a rat DHFR and may be introduced into aparent mouse cell line which expresses the mouse DHFR). The amplifiablegene (or marker) and the selectable marker may be present on the samevector; alternatively, they may be present on two separate vectors.

In one embodiment the second inhibitable enzyme expressed by the parentcell line is selected from the group consisting of dihydrofolatereductase, glutamine synthetase, adenosine deaminase, asparaginesynthetase.

In another embodiment, the method of the present invention theconcentration of inhibitor present in the first aqueous solution (e.g.,tissue culture medium) used to allow identification of the transformedcell(s) containing amplified copies of the amplification vector andamplified copies of the expression vector(s) is four-fold to six-foldthe concentration required to prevent the growth of the parent cellline. It is well understood by those skilled in the art that only thosesequences present on the amplification vector and expression vector(s)which are required for the expression of the inhibitable enzyme and theprotein(s) of interest, respectively, need to be amplified. However, itis also well understood that any vector backbone sequences linked to thesequences required for expression of the inhibitable enzyme orprotein(s) of interest may also be amplified (and typically are) duringthe co-amplification process.

In still another embodiment, the method of the present invention furthercomprises providing a selection vector encoding a selectable geneproduct (ie., a selectable marker) which is introduced into said parentcell line together with said expression vector and said amplificationvector (alternatively, the selectable marker may be present on the samevector which contains the amplifiable marker). The invention is notlimited by the nature of the selectable gene product employed. Theselectable gene product employed may be a dominant selectable markerincluding but not limited to hygromycin G phosphotransferase (e.g., thehyg gene product), xanthine-guanine phosphoribosyltransferase (e.g., thegpt gene product) and aminoglycoside 3' phosphotransferase (e.g., theneo gene product). Alternatively, the selectable marker employed mayrequire the use of a parent cell line which lacks the enzymatic activityencoded by the selectable marker such as hypoxanthine guaninephosphoribosyltransferase, thymidine kinase or carbamoyl-phosphatesynthetase-aspartate transcarbamoylase-dihydrooratase. In a particularlypreferred embodiment, the selection vector encodes an activehypoxanthine guanine phosphoribosyltransferase. When the selectionvector encodes an active hypoxanthine guanine phosphoribosyltransferase,the second aqueous solution which requires the expression of thisselectable gene product comprises hypoxanthine and azaserine.

In another embodiment, the method of the present invention furthercomprises following the introduction of the vectors (ie., theamplification, expression and selection vectors), the additional step ofgrowing the transformed cell in a second aqueous solution which requiresthe expression of the selectable gene product prior to growing thetransformed cell in a first aqueous solution containing an inhibitorcapable of inhibiting said inhibitable enzyme.

The method of the present invention is not limited by the nature of theinhibitable enzyme encoded by the amplification vector; the art is wellof aware of numerous amplifiable markers. In a preferred embodiment, theamplification vector encodes an active enzyme selected from the groupconsisting of dihydrofolate reductase, glutamine synthetase, adenosinedeaminase, asparagine synthetase.

In another preferred embodiment, the inhibitor used to select for atransformed cell expressing the inhibitable enzyme encoded by theamplification vector is selected from the group consisting ofmethotrexate, 2'-deoxycoformycin, methionine sulphoximine, albizziin andβ-aspartyl hydroxamate.

The present invention further provides a method of treating B-celllymphoma, comprising: a) providing: i) a subject having a B-celllymphoma; ii) a multivalent vaccine comprising at least two recombinantvariable regions of immunoglobulin molecules derived from the subjects'sB-cell lymphoma cells, wherein the cells express at least two differentimmunoglobulin molecules, the immunoglobulin molecules differing by atleast one idiotope; b) administering said multivalent vaccine to thesubject. In a preferred embodiment, the vaccine comprises at least tworecombinant immunoglobulin molecules comprising the recombinant variableregions derived from the lymphoma cells. In a preferred embodiment, themethod employs a multivalent vaccine which further comprises anadjuvant. When the vaccine is to be administered to a human subject,adjuvants approved for use in humans are employed. In a preferredembodiment the adjuvant is Syntex adjuvant formulation 1. Therecombinant Ig proteins (including fragments of Ig proteins) whichcomprise the multivalent vaccine may be conjugated to a carrier proteinsuch as KLH.

The present invention provides a method of treating B-cell lymphoma,comprising: a) providing: i) a subject having a B-cell lymphoma; ii) amultivalent vaccine comprising at least two recombinant variable regionsof immunoglobulin molecules derived from the subjects's B-cell lymphomacells, wherein the cells express at least two different immunoglobulinmolecules, the immunoglobulin molecules differing by at least oneidiotope; and iii) dendritic cells isolated from the subject; b)incubating the dendritic cells in vitro with the multivalent vaccine toproduce autologous antigen-pulsed dendritic cells; c) administeringintravenously the pulsed dendritic cells to the subject; and d)following the administration of the pulsed dendritic cells,administering the multivalent vaccine to the subject. In a preferredembodiment, the vaccine comprises at least two recombinantimmunoglobulin molecules comprising the recombinant variable regions.

The present invention further provides a method of treating B-celllymphoma, comprising: a) providing: i) a subject having a B-celllymphoma; ii) a vaccine produced according to the methods of the presentinvention; and b) administering the vaccine to the subject.

Still further, the present invention provides a method of treating asubject having an immune cell tumor, comprising: a) providing: i) immunecell tumor cells isolated from a subject, the tumor cells expressing anidiotype protein on the cell membrane; ii) an amplification vectorcomprising a first recombinant oligonucleotide having a sequenceencoding a first inhibitable enzyme operably linked to a heterologouspromoter; iii) a eukaryotic parent cell line; b) isolating nucleotidesequences encoding at least one idiotype protein expressed on thesurface of the tumor cells; c) inserting the nucleotide sequencesencoding the idiotype protein(s) into at least one vector to produce atleast one expression vector capable of expressing the idiotypeprotein(s); d) introducing the expression vector(s) into the parent cellto generate one or more transformed cells; e) growing the transformedcell in a first aqueous solution containing an inhibitor capable ofinhibiting the inhibitable enzyme wherein the concentration of theinhibitor present in the first aqueous solution is sufficient to preventgrowth of the parent cell line; f) identifying a transformed cellcapable of growth in the first aqueous solution, wherein the transformedcell capable of growth contains an amplified number of copies of theexpression vector and an amplified number of copies of the amplificationvector and wherein the transformed cell produces the idiotype protein(s)encoded by the expression vector(s); g) isolating the idiotypeprotein(s) produced by the transformed cell; and h) administering theisolated idiotype protein(s) to the subject.

The method of the present invention is not limited by the nature of thetumor cells. In one embodiment, the tumor cells are T lymphoid cells andthe idiotype protein is a T cell receptor or fragment thereof. Inanother embodiment, the tumor cells are B lymphoid cells and theidiotype protein is an immunoglobulin or fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the map of the expression vector pSSD5. Selectedrestriction enzyme sites are indicated.

FIG. 2 shows the map of the expression vector pSSD7. Selectedrestriction enzyme sites are indicated.

FIG. 3 shows the map of the expression vector pSRαSD5. Selectedrestriction enzyme sites are indicated.

FIG. 4 shows the map of the expression vector pSRαSD7. Selectedrestriction enzyme sites are indicated.

FIG. 5 shows the map of the expression vector pMSD5. Selectedrestriction enzyme sites are indicated.

FIG. 6 shows the map of the expression vector pMSD7. Selectedrestriction enzyme sites are indicated.

FIG. 7 shows the map of the expression vector pHEF1αASD5. Selectedrestriction enzyme sites are indicated.

FIG. 8 shows the map of the expression vector pHEF1αASD7. Selectedrestriction enzyme sites are indicated.

FIG. 9 shows the map of the expression vector pHEF1αBSD5. Selectedrestriction enzyme sites are indicated.

FIG. 10 shows the map of the expression vector pHEF1αBSD7. Selectedrestriction enzyme sites are indicated.

FIG. 11 shows the map of the expression vector pMSD5-HPRT. Selectedrestriction enzyme sites are indicated.

FIG. 12 shows the map of the expression vector pSSD7-DHFR. Selectedrestriction enzyme sites are indicated.

FIG. 13 shows the map of the expression vector pJFE 14. Selectedrestriction enzyme sites are indicated.

FIG. 14 shows the map of the expression vector pJFE 14ΔIL10. Selectedrestriction enzyme sites are indicated.

FIG. 15 shows the map of the expression vector pSRαSD-DRα-DAF. Selectedrestriction enzyme sites are indicated.

FIG. 16 shows the map of the expression vector pSRαSD-DRβ1-DAF. Selectedrestriction enzyme sites are indicated.

FIG. 17 is a histogram showing the clone 5 cells selected for growth inhypoxanthine and azaserine stained with the L243 monoclonal antibody.

FIG. 18 is a histogram showing the clone 5 cells selected for growth in80 nM MTX stained with the L243 monoclonal antibody.

FIG. 19 is a histogram showing the clone 5 cells selected for growth in320 nM MTX stained with the L243 monoclonal antibody.

FIG. 20 is a histogram showing the clone 5 cells selected for growth in1 μM MTX stained with the L243 monoclonal antibody.

FIG. 21 shows the map of the expression vector pSRαSD9. Selectedrestriction enzyme sites are indicated.

FIG. 22 shows the map of the expression vector pSRαSD9CG3C. Selectedrestriction enzyme sites are indicated.

FIG. 23 shows the map of the expression vector pSRαSD9CG4C. Selectedrestriction enzyme sites are indicated.

FIG. 24 shows the map of the expression vector pSRαSDCKC. Selectedrestriction enzyme sites are indicated.

FIG. 25 shows the map of the expression vector pSRαSDCL2C. Selectedrestriction enzyme sites are indicated.

FIG. 26 shows the map of the selection and amplification vectorpM-HPRT-SSD9-DHFR. Selected restriction enzyme sites are indicated.

DEFINTIONS

To facilitate understanding of the invention, a number of terms aredefined below.

The term "recombinant DNA molecule" as used herein refers to a DNAmolecule which is comprised of segments of DNA joined together by meansof molecular biological techniques.

The terms "in operable combination" or "operably linked" as used hereinrefers to the linkage of nucleic acid sequences in such a manner that anucleic acid molecule capable of directing the synthesis of a desiredprotein molecule is produced. When a promoter sequence is operablylinked to sequences encoding a protein, the promoter directs theexpression of mRNA which can be translated to produce a functional formof the encoded protein. The term also refers to the linkage of aminoacid sequences in such a manner that a functional protein is produced.

DNA molecules are said to have "5' ends" and "3' ends" becausemononucleotides are reacted to make oligonucleotides in a manner suchthat the 5' phosphate of one mononucleotide pentose ring is attached tothe 3' oxygen of its neighbor in one direction via a phosphodiesterlinkage. Therefore, an end of an oligonucleotides is referred to as the"5' end" if its 5' phosphate is not linked to the 3' oxygen of amononucleotide pentose ring and as the "3' end" if its 3' oxygen is notlinked to a 5' phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5' and 3' ends. In either alinear or circular DNA molecule, discrete elements are referred to asbeing "upstream" or 5' of the "downstream" or 3' elements. Thisterminology reflects the fact that transcription proceeds in a 5' to 3'fashion along the DNA strand. The promoter and enhancer elements whichdirect transcription of a linked gene are generally located 5' orupstream of the coding region (enhancer elements can exert their effecteven when located 3' of the promoter element and the coding region).Transcription termination and polyadenylation signals are located 3'ordownstream of the coding region.

The term "an oligonucleotide having a nucleotide sequence encoding agene" means a DNA sequence comprising the coding region of a gene or, inother words, the DNA sequence which encodes a gene product. The codingregion may be present in either a cDNA or genomic DNA form. Suitablecontrol elements such as enhancers/promoters, splice junctions,polyadenylation signals, etc. may be placed in close proximity to thecoding region of the gene if needed to permit proper initiation oftranscription and/or correct processing of the primary RNA transcript.Alternatively, the coding region utilized in the expression vectors ofthe present invention may contain endogenous enhancers/promoters, splicejunctions, intervening sequences, polyadenylation signals, etc. or acombination of both endogenous and exogenous control elements.

The term "recombinant oligonucleotide" refers to an oligonucleotidecreated using molecular biological manipulations, including but notlimited to, the ligation of two or more oligonucleotide sequencesgenerated by restriction enzyme digestion of a polynucleotide sequence,the synthesis of oligonucleotides (e.g., the synthesis of primers oroligonucleotides) and the like.

The term "recombinant oligonucleotide having a sequence encoding aprotein operably linked to a heterologous promoter" or grammaticalequivalents indicates that the coding region encoding the protein (e.g.,an enzyme) has been joined to a promoter which is not the promoternaturally associated with the coding region in the genome of an organism(i.e., it is linked to an exogenous promoter). The promoter which isnaturally associated or linked to a coding region in the genome isreferred to as the "endogenous promoter" for that coding region.

The term "transcription unit" as used herein refers to the segment ofDNA between the sites of initiation and termination of transcription andthe regulatory elements necessary for the efficient initiation andtermination. For example, a segment of DNA comprising anenhancer/promoter, a coding region, and a termination andpolyadenylation sequence comprises a transcription unit.

The term "regulatory element" as used herein refers to a genetic elementwhich controls some aspect of the expression of nucleic acid sequences.For example, a promoter is a regulatory element which facilitates theinitiation of transcription of an operably linked coding region. Otherregulatory elements are splicing signals, polyadenylation signals,termination signals, etc. (defined infra).

The term "expression vector" as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Nucleic acid sequencesnecessary for expression in prokaryotes include a promoter, optionallyan operator sequence, a ribosome binding site and possibly othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

Transcriptional control signals in eucaryotes comprise "promoter" and"enhancer" elements. Promoters and enhancers consist of short arrays ofDNA sequences that interact specifically with cellular proteins involvedin transcription [Maniatis, et al., Science 236:1237 (1987)]. Promoterand enhancer elements have been isolated from a variety of eukaryoticsources including genes in yeast, insect and mammalian cells and viruses(analogous control elements, ie., promoters, are also found inprokaryotes). The selection of a particular promoter and enhancerdepends on what cell type is to be used to express the protein ofinterest. Some eukaryotic promoters and enhancers have a broad hostrange while others are functional in a limited subset of cell types [forreview see Voss, et al., Trends Biochem. Sci., 11:287 (1986) andManiatis, et al., supra (1987)]. For example, the SV40 early geneenhancer is very active in a wide variety of cell types from manymammalian species and has been widely used for the expression ofproteins in mammalian cells [Dijkema, et al., EMBO J. 4:761 (1985)]. Twoother examples of promoter/enhancer elements active in a broad range ofmammalian cell types are those from the human elongation factor 1α gene[Uetsuki et al., J. Biol. Chem., 264:5791 (1989); Kim et al., Gene91:217 (1990); and Mizushima and Nagata, Nuc. Acids. Res., 18:5322(1990)] and the long terminal repeats of the Rous sarcoma virus [Gormanet al., Proc. Natl. Acad. Sci. USA 79:6777 (1982)] and the humancytomegalovirus [Boshart et al., Cell 41:521 (1985)].

The term "promoter/enhancer" denotes a segment of DNA which containssequences capable of providing both promoter and enhancer functions (forexample, the long terminal repeats of retroviruses contain both promoterand enhancer functions). The enhancer/promoter may be "endogenous" or"exogenous" or "heterologous." An endogenous enhancer/promoter is onewhich is naturally linked with a given gene in the genome. An exogenous(heterologous) enhancer/promoter is one which is placed in juxtapositionto a gene by means of genetic manipulation (ie., molecular biologicaltechniques).

The presence of "splicing signals" on an expression vector often resultsin higher levels of expression of the recombinant transcript. Splicingsignals mediate the removal of introns from the primary RNA transcriptand consist of a splice donor and acceptor site [Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, New York (1989) pp. 16.7-16.8]. A commonly used splicedonor and acceptor site is the splice junction from the 16S RNA of SV40.

Efficient expression of recombinant DNA sequences in eukaryotic cellsrequires signals directing the efficient termination and polyadenylationof the resulting transcript. Transcription termination signals aregenerally found downstream of the polyadenylation signal and are a fewhundred nucleotides in length. The term "poly A site" or "poly Asequence" as used herein denotes a DNA sequence which directs both thetermination and polyadenylation of the nascent RNA transcript. Efficientpolyadenylation of the recombinant transcript is desirable astranscripts lacking a poly A tail are unstable and are rapidly degraded.The poly A signal utilized in an expression vector may be "heterologous"or "endogenous." An endogenous poly A signal is one that is foundnaturally at the 3' end of the coding region of a given gene in thegenome. A heterologous poly A signal is one which is isolated from onegene and placed 3' of another gene. A commonly used heterologous poly Asignal is the SV40 poly A signal. The SV40 poly A signal is contained ona 237 bp BamHI/BclI restriction fragment and directs both terminationand polyadenylation [Sambrook, supra, at 16.6-16.7]. This 237 bpfragment is contained within a 671 bp BamHI/PstI restriction fragment.

The term "stable transfection" or "stably transfected" refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term "stable transfectant" refers to a cell whichhas stably integrated foreign DNA into the genomic DNA.

The term "stable transfection" or "stably transfected" refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term "stable transfectant" refers to a cell whichhas stably integrated foreign or exogenous DNA into the genomic DNA ofthe transfected cell.

The terms "selectable marker" or "selectable gene product" as usedherein refer to the use of a gene which encodes an enzymatic activitythat confers resistance to an antibiotic or drug upon the cell in whichthe selectable marker is expressed. Selectable markers may be"dominant"; a dominant selectable marker encodes an enzymatic activitywhich can be detected in any mammalian cell line. Examples of dominantselectable markers include the bacterial aminoglycoside 3'phosphotransferase gene (also referred to as the neo gene) which confersresistance to the drug G418 in mammalian cells, the bacterial hygromycinG phosphotransferase (hyg) gene which confers resistance to theantibiotic hygromycin and the bacterial xanthine-guanine phosphoribosyltransferase gene (also referred to as the gpt gene) which confers theability to grow in the presence of mycophenolic acid. Other selectablemarkers are not dominant in that their use must be in conjunction with acell line that lacks the relevant enzyme activity. Examples ofnon-dominant selectable markers include the thymidine kinase (tk) genewhich is used in conjunction with TK⁻ cell lines, thecarbamoyl-phosphate synthetase-aspartatetranscarbamoylase-dihydroorotase (CAD) gene which is used in conjunctionwith CAD-deficient cells and the mammalian hypoxanthine-guaninephosphoribosyl transferase (hprt) gene which is used in conjunction withHPRT⁻ cell lines. A review of the use of selectable markers in mammaliancell lines is provided in Sambrook et al., supra at pp.16.9-16.15. It isnoted that some selectable markers can be amplified and therefore can beused as amplifiable markers (e.g., the CAD gene).

The term "amplification" or "gene amplification" as used herein refersto a process by which specific DNA sequences are disproportionatelyreplicated such that the amplified gene becomes present in a higher copynumber than was initially present in the genome. Gene amplificationoccurs naturally during development in particular genes such as theamplification of ribosomal genes in amphibian oocytes. Geneamplification may be induced by treating cultured cells with drugs. Anexample of drug-induced amplification is the methotrexate-inducedamplification of the endogenous dhfr gene in mammalian cells [Schmike etaL (1978) Science 202:1051]. Selection of cells by growth in thepresence of a drug (e.g., an inhibitor of an inhibitable enzyme) mayresult in the amplification of either the endogenous gene encoding thegene product required for growth in the presence of the drug or byamplification of exogenous (i.e., input) sequences encoding this geneproduct, or both.

The term "co-amplification" as used herein refers to the introductioninto a single cell of an amplifiable marker in conjunction with othergene sequences (comprising one or more non-selectable genes such asthose contained within an expression vector) and the application ofappropriate selective pressure such that the cell amplifies both theamplifiable marker and the other, non-selectable gene sequences. Theamplifiable marker may be physically linked to the other gene sequencesor alternatively two separate pieces of DNA, one containing theamplifiable marker and the other containing the non-selectable marker,may be introduced into the same cell.

The termt "amplifiable marker," "amplifiable gene" or "amplificationvector" is used herein to refer to a gene or a vector encoding a genewhich permits the amplification of that gene under appropriate growthconditions. Vectors encoding the dihydrofolate reductase (dhfr) gene canbe introduced into appropriate cell lines (typically a dhfr⁻ cell) andgrown in the presence of increasing concentrations of the DHFR inhibitormethotrexate to select for cells which have amplified the dhfr gene. Theadenosine deaminase (ada) gene has been used in analogous fashion toallow the amplification of ada gene sequences in cells selected forgrowth in the presence of ADA inhibitors such as 2'-deoxycoformycin.Examples of other genes which can be used as amplifiable markers inmammalian cells include the CAD gene (inhibitor:N-phosphonoacetyl-L-aspartic acid), the ornithine decarboxylase gene(inhibitor: difluoromethylornithine in medium lacking putrescine), andthe asparagine synthetase gene (inhibitors: albizziin or β-aspartylhydroxamate in asparagine-free medium) [see Kaufman, Methods inEnzymol., 185:537 (1990) for a review].

The term "gene of interest" as used herein refers to the gene insertedinto the polylinker of an expression vector whose expression in the cellis desired for the purpose of performing further studies on thetransfected cell. The gene of interest may encode any protein whoseexpression is desired in the transfected cell at high levels. The geneof interest is not limited to the examples provided herein; the gene ofinterest may include cell surface proteins, secreted proteins, ionchannels, cytoplasmic proteins, nuclear proteins (e.g., regulatoryproteins), mitochondrial proteins, etc.

The terms "nucleic acid molecule encoding," "DNA sequence encoding," and"DNA encoding" refer to the order or sequence of deoxyribonucleotidesalong a strand of deoxyribonucleic acid. The order of thesedeoxyribonucleotides determines the order of amino acids along thepolypeptide (protein) chain. The DNA sequence thus codes for the aminoacid sequence.

The vertebrate hematopoietic system comprises cells of the lymphoid andmyeloid lineages. The myeloid lineage (or myeloid-erythroid lineage)gives rise to erythrocytes, basophils, neutrophils, macrophages,eosinophils and platelets. The lymphoid lineage gives rise to Blymphocytes, including plasma cells, and T lymphocytes.

The term "lymphoid" when used in reference to a cell line or a cell,means that the cell line or cell is derived from the lymphoid lineageand includes cells of both the B and the T lymphocyte lineages.

The terms "T lymphocyte" and "T cell" as used herein encompass any cellwithin the T lymphocyte lineage from T cell precursors (including Thy1positive cells which have not rearranged the T cell receptor genes) tomature T cells (i.e., single positive for either CD4 or CD8, surface TCRpositive cells).

The terms "B lymphocyte" and "B cell" encompasses any cell within the Bcell lineage from B cell precursors, such as pre-B cells (B220⁺ cellswhich have begun to rearrange Ig heavy chain genes), to mature B cellsand plasma cells. "Myeloma" cells or cell lines are malignant plasmacells or cell lines (and are thus in the B cell lineage, not the T celllineage).

The terms "parent cell line" or "parental cell line" refers to a cellline prior to the addition of exogenous nucleic acid.

The term "transformed cells" refers to cells which contain exogenous DNA(i.e., heterologous DNA introduced into the cells such as theintroduction of an expression vector). Terms "transformed cell" and"transfected cell" are used herein interchangeably.

The term "aqueous solution" when used in reference to a solution used togrow a cell line refers to a solution containing compounds required tosupport the growth of the cells and may contain salts, buffering agents,serum or synthetic serum replacements. An aqueous solution capable ofsupporting the growth of a cell line is also referred to as "tissueculture medium" (e.g., EMEM, DMEM, RMPI 1470, etc.).

An "aqueous solution which requires the expression of a selectable geneproduct" is a solution or tissue culture medium which forces a cell lineto express a function or active form of the selectable gene product inorder for the cells to survive in this medium (e.g., the cell mustexpress a functional HPRT when grown in medium containing hypoxanthineand azaserine). "Aqueous solutions which contain an inhibitor capable ofinhibiting an inhibitable enzyme" expressed by a cell refers to mediumcontaining an inhibitor (e.g., methotrexate) which is capable ofinhibiting an inhibitable enzyme (e.g., DHFR). The presence of theinhibitor in the medium requires the cell to express a functional oractive form of the enzyme which is inhibited by the inhibitor in orderto survive.

The "concentration of an inhibitor sufficient to prevent the growth ofthe parent cell line" refers to that concentration of inhibitor whichmust be present in the medium to achieve the killing of greater than 98%of the cells within 3 to 5 days after plating the parent cells in mediumcontaining the inhibitor.

The term "amplified number of copies of a vector" refers to a cell linewhich has incorporated an exogenous or recombinant vector and hasincreased the number of copies of the vector present in the cell byvirtue of the process of gene amplification.

The term "amplified gene" refers to a gene present in multiple copies ina cell line by virtue of gene amplification.

A cell which contains an "endogenous gene encoding an inhibitableenzyme" refers to cell which naturally (as opposed to by virtue ofrecombinant DNA manipulations) contains in its genomic DNA a geneencoding an inhibitable enzyme; the coding region of this gene will beoperably linked to and under the control of its endogenous promoter.

The term "active enzyme" refers to an enzyme which is functional (ie.,capable of carrying out the enzymatic function).

Immunoglobulin molecules consist of heavy (H) and light (L) chains,which comprise highly specific variable regions at their amino termini.The variable (V) regions of the H (V_(H)) and L (V_(L)) chains combineto form the unique antigen recognition or antigen combining site of theimmunoglobulin (Ig) protein. The variable regions of an Ig moleculecontain determinants (i.e., molecular shapes) that can be recognized asantigens or idiotypes.

The term "idiotype" refers to the set of antigenic or epitopicdeterminants (i.e., idiotopes) of an immunoglobulin V domain (i.e., theantigen combining site formed by the association of the complementaritydetermining regions or V_(H) and V_(L) regions).

The term "idiotope" refers to a single idiotypic epitope located along aportion of the V region of an immunoglobulin molecule.

The term "anti-idiotypic antibody" or grammatical equivalents refers toan antibody directed against a set of idiotopes on the V region of an Igprotein.

A "multivalent vaccine" when used in reference to a vaccine comprisingan idiotypic protein or fragment thereof (e.g., immunoglobulin moleculesor variable regions thereof, T cell receptor proteins or variableregions thereof) refers to a vaccine which contains at least twoidiotypic proteins which differ by at least one idiotope. For example, avaccine which contains two or more immunoglobulin molecules derived froma B-cell lymphoma where the immunoglobulin molecules differ from oneanother by at least one idiotope (e.g., these immunoglobulins aresomatic variants of one another) is a multivalent vaccine.

As used herein "recombinant variable regions of immunoglobulinmolecules" refers to variable regions of Ig molecules which are producedby molecular biological means. As shown herein, the variable domain ofthe heavy and light chains may be molecularly cloned from lymphoma cellsand expressed in a host cell (e.g., by insertion into an expressionvector followed by transfer of the expression vector into a host cell);variable domains expressed in this manner are recombinant variableregions of immunoglobulin molecules. The recombinant variable regions ofimmunoglobulin molecules may be expressed as an immunoglobulin moleculecomprising the recombinant variable regions operably linked to theappropriate constant region (i.e., C_(H) or C_(L)) (the constant regionmay comprise the constant region naturally associated with therecombinant variable region, as a Fab, F(ab')₂ or Fab' fragmentcomprising the variable domain of the heavy and light chains, theconstant region of the light chain and a portion of the constant regionof the heavy chain (the Fab, F(ab')₂ or Fab' fragments may be created bydigestion of a recombinant immunoglobulin molecule or alternatively,they may be produced by molecular biological means), or alternatively,as a single chain antibody or Fv protein.

"Single-chain antibodies" or "Fv" consist of an antibody light chainvariable domain or region ("V_(L) ") and heavy chain variable region("V_(H) ") connected by a short peptide linker. The peptide linkerallows the structure to assume a conformation which is capable ofbinding to antigen [Bird et al., (1988) Science 242:423 and Huston etal. (1988) Proc. Natl. Acad. Sci. USA 85:5879].

A "recombinant variable region derived from a lymphoma cell" refers to avariable region which is molecularly cloned from RNA isolated from alymphoma cell. The recombinant variable domain may be expressed as anentire immunoglobulin molecule or may be expressed as a fragment of animmunoglobulin molecule, including Fv molecules.

An "immune-enhancing cytokine" is a cytokine that is capable ofenhancing the immune response when the cytokine is generated in situ oris administered to a mammalian host. Immune-enhancing cytokine include,but are not limited to, granulocyte-macrophage colony stimulating factor(G-CSF), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4(IL-4) and interleukin-12 (IL-12).

An "adjuvant" is a compound which enhances or stimulates the immuneresponse when administered with an antigen(s).

"Malignant cells isolated from a patient having a B-cell lymphoma"refers to the malignant or pathogenic B-cells found within the solidtumors characteristic of lymphoma (e.g., lymph nodes and spleencontaining the tumor cells).

DESCRIPTION OF THE INVENTION

The invention provides vectors and improved methods for the expressionand co-amplification of genes encoding recombinant proteins in culturedcells. The description is divided into the following sections: I)Overview of Co-Amplification Methods; II) Expression Vectors; III)Amplification Vectors; IV) Selection Vectors; V) Cell Lines and CellCulture; VI) Co-Transfection of Cell Lines; VII) Selection andCo-Amplification; VIII) Co-Amplification Without Prior Selection; IX)High-Level Expression of Interleukin 10 in Amplified Cell Lines; X)High-Level Expression of Human Class II MHC Antigens and T Cell ReceptorProteins in Amplified Cell Lines; and XI) Production of CustomMultivalent Vaccines For the Treatment of Lymphoma and Leukemia.

I. Overview of Co-Amplification Methods

The present invention provides improved methods for the co-amplificationof selectable and non-selectable genes in eukaryotic cell lines. Thepresent invention allows, for the first time, the co-amplification ofrecombinant gene sequences in T lymphoid cell lines (e.g., theBW5147.G.1.4 cell line).

The ability to amplify gene sequences in lymphoid cell lines (T or Blymphoid lines) is desirable for a number of reasons. These include theability to of these cells to secrete recombinant immunoglobulins and theability to grow these suspension cell lines at high biomass infermentators. To date amplification of input gene sequences has beenreported only in B lymphoid cell lines (e.g., myeloma cell lines).Further, the ability to amplify genes in myeloma cell lines using thedhfr gene as the amplifiable marker have been problematic due to theendogenous DHFR activity in the myeloma cells. Successful amplificationis reported to require the use of a MTX-resistant dhfr gene and the useof very high levels of MTX [Dorai and Moore (1987) J. Immunol.139:4232]. In contrast, the present invention does not require the useof a MTX-resistant dhfr gene and permits the amplification of genes in Tlymphoid cell lines.

A co-amplification scheme employing the glutamine synthetase (GS) genehas been described [U.S. Pat. No. 5,122,464, the disclosure of which isincorporated by reference herein and Bebbington, et al. (1992)Bio/Technology 10:169]. This co-amplification scheme was developed inpart to circumvent the need to use very high levels of MTX and aMTX-resistant dhfr gene to achieve co-amplification of genes in myelomacells. The use of GS in co-amplification schemes has several drawbacks.First, the propensity of the endogenous GS locus in each cell line to beused must be examined to preclude the use of cell lines in which theendogenous GS locus will amplify at a frequency which makes the GS geneusable. Of four myeloma or hybridoma cell lines, examined, two of thefour (50%) were found to be unsuitable host cells for the use of GS as aselectable marker (Bebbington, et al., supra). One of these twounsuitable cell lines, SP2/0, was found to amplify the endogenous GSlocus.

A second drawback to the use of GS as a selectable and amplifiablemarker is the amount of time required for the isolation of cell linesproducing high levels of the non-selected gene product. A single roundof amplification and recloning was reported to require 3 months using amyeloma cell line subjected to GS selection (Bebbington, et al., supra).Other selectable markers used in co-amplification protocols have beenreported to require even longer periods of time; selection of amplifiedmyeloma cell lines using DHFR as the selectable marker takes up to 6months [Dorai and Moore (1987) J. Immunol. 139:4232].

The present invention provides methods which allow the isolation of thedesired amplified cell lines in a shorter period of time than permittedusing existing co-amplification protocols. Multiple rounds ofamplification can be achieved using the present invention in a period ofabout 3 months. The savings in time is realized, in part, by the use ofcell lines which have rapid doubling times as the host cell line. Inaddition to shortening the period required for the generation of thedesired amplified cell line, the present methods generate with highfrequency amplified cell lines which have co-amplified thenon-selectable gene(s) of interest as well as the amplifiable gene(e.g., the dhfr gene).

In general the present invention involves the following steps:

1. Introduction of linearized plasmids comprising an expressionvector(s) encoding a protein of interest, an amplification vectorencoding an amplifiable marker (e.g., the dhfr gene) and, optionally, aselection vector encoding a selectable marker (e.g., HPRT) into a hostcell line. The host cell line will have a doubling time of 12 hours orless; a particularly preferred host cell line is the BW5147.G.1.4 cellline. The host cell prior to the introduction of the linearized vectorsis referred to as the parental cell line. A preferred means ofintroducing the vector DNA into the host cell line is electroporation.The ratio of the amplification vector, non-selectable expressionvector(s) and/or selection vector is important. A ratio of 1 (selectablevector): 2 (amplification vector): 20-25 [expression vector(s)] isemployed. If a selectable marker is not employed a ratio of 1(amplification vector): 10-15 [expression vector(s)] is used. The use ofthis ratio in conjunction with the electroporation of linearized vectorDNA produces random concatemers of the transfected DNA vectors whichcontain a low percentage of the amplifiable gene. While not limiting theinvention to any particular mechanism, it is believed that these randomconcatemers containing a low percentage of the amplifiable gene are lesslikely to generate an amplification unit composed primarily of theamplifiable marker. It is desirable to produce an amplification unitwhich contains primarily the expression vector(s) as this results in anamplified cell line which is expressing large quantities of theprotein(s) of interest.

In contrast to existing transfection methods (including electroporationprotocols), the methods of the present invention employ large quantitiesof DNA comprising the gene(s) of interest (i.e., the expression vector)[for a discussion of current electroporation methods see Ausubel et al.,Current Protocols in Molecular Biology (1995) John Wiley & Sons, Inc.,at 9.3.1 to 9.3.6]. Using the methods of the present invention, a totalof about 500 to 750 μg of DNA comprising the expression vector(s), theamplification vector and if employed, the selection vector in a totalvolume of 0.5 ml are introduced into approximately 2×10⁷ cells in 0.5 ofthe electroporation buffer (final density of DNA is therefore 1 to 1.5mg/ml). The use of large quantities of the expression vectors increasesthe frequency with which clones of cells expressing the gene productsencoded by the exogenous DNA are isolated. Using the methods of thepresent invention about 20 to 25% of the selectants (or primaryamplificants if no selection vector is employed) express the genes ofinterest at relatively high levels. In contrast, using conventionalamounts of DNA (about 20 to 40 μg when introducing a single expressionvector into the cells), only 1 to 5% of the selectants isolated expressthe gene of interest at relatively high levels.

2. When a selection vector is employed, the transfected cells areallowed to recover by growth in their normal growth medium for a shortperiod (about 36 to 48 hours) and then they are placed in medium whichrequires the cells to express the selectable marker in order to survive(selective medium). The use of the selective medium facilitates theidentification of cells which have taken up the transfected DNA.Colonies of cells which grow in the selective medium (selectants) areexpanded and examined for the ability to express the protein ofinterest. Selectant clones which express the protein(s) of interest athigh levels are then subjected to the amplification process.

3. Selectant clones expressing the protein(s) of interest at high levelsare examined to determine their level of sensitivity to the inhibitorwhich inhibits the enzyme encoded by the amplifiable vector. Thesensitivity of the parental cell line to the inhibitor is alsodetermined. Selectants which survive growth in medium containing up to a6-fold higher concentration (typically 4- to 6-fold higher) of theinhibitor than that required to kill the parental cell line are selectedfor further manipulation (the first round amplificants). [Any primarytransfectant which has clearly taken up a transfected amplificationvector (e.g., one encoding DHFR) is suitable for continuation with theamplification protocols of the present invention. The presence of thetransfected amplification vector is indicated by the ability of theprimary transfectant to grow in medium containing the inhibitor at alevel which is above the level required to kill the parental cell line.]The first round amplificants are examined for the expression of theprotein(s) of interest. Cells which express low levels of the protein ofinterest are discarded (as this indicates a lack of co-ordinateamplification of the amplifiable gene and the gene(s) of interest).Selectants which are capable of growing in medium containing greaterthan 6-fold the concentration of inhibitor which prevents the growth ofthe parental cell line are discarded. It has been found that selectantswhich are resistant to extremely high levels of the inhibitor typicallydo not yield amplified cell lines which express high quantities of theprotein of interest. While not limiting the present invention to anyparticular mechanism, it is thought that resistance to extremely highlevels of inhibitor at the first round of amplification is indicative ofa cell line in which the amplifiable gene sequences readily separateaway from the majority of the other input DNA sequences (e.g., theexpression vector) resulting the amplification of an amplified unitcomprising primarily the amplifiable gene sequences.

4. The first round amplificants which are capable of growing in mediumcontaining 4-fold to 6-fold higher concentrations of the inhibitor thanthat required to kill the parental cell line are grown in mediumcontaining this level of inhibitor for 2 to 3 weeks. The cells are thengrown in medium containing about 4- to 6-fold more of the inhibitor(i.e., 16- to 36-fold the concentration which kills the parental cells)to generate the second round amplificants. The level of expression ofthe protein(s) of interest are examined in the second roundamplificants; any clones which do not show an increase in expression ofthe protein(s) of interest which corresponds with the increasedresistance to the inhibitor are discarded.

5. The amplified cell lines are subjected to subsequent rounds ofamplification by increasing the level of inhibitor in the medium 4- to6-fold for each additional round of amplification. At each round ofamplification, the expression of the protein(s) of interest is examined.Typically any discordance between the level of resistance to theinhibitor and the level of expression of the protein(s) if interest isseen on the second round of amplification. Using the methods of thepresent invention more than 60% of the first round amplificants willco-amplify the gene(s) of interest and the amplifiable gene in thesecond round of amplification. All clones which co-amplified the gene(s)of interest and the amplifiable gene in the second round ofamplification have been found to continue to coordinately amplify thesegene sequences in all subsequent rounds of amplification until a maximumexpression level was reached.

The following provides additional details regarding the various stepsand components employed in the co-amplification protocols of the presentinvention.

II. Expression Vectors

The expression vectors of the invention comprise a number of geneticelements: A) a plasmid backbone; B) regulatory elements which permit theefficient expression of genes in eukaryotic cells--these includeenhancer/promoter elements, poly A signals and splice junctions; C)polylinkers which allow for the easy insertion of a gene (a selectablemarker gene, an amplifiable marker gene or a gene of interest) into theexpression vector; and D) constructs showing the possible combination ofthe genetic elements. These genetic elements may be present on theexpression vector in a number of configurations and combinations.

A. Plasmid Backbone

The expression vector contains plasmid sequences which allow for thepropagation and selection of the vector in procaryotic cells; theseplasmid sequences are referred to as the plasmid backbone of the vector.While not intending to limit the invention to a particular plasmid, thefollowing plasmids are preferred. The pUC series of plasmids and theirderivatives which contain a bacterial origin of replication (the pMB1replicon) and the β-lactamase or ampicillin resistance gene. The pUCplasmids, such as pUC18 (ATCC 37253) and pUC19 (ATCC 37254), areparticularly preferred as they are expressed at high copy number(500-700) in bacterial hosts. pBR322 and its derivatives which containthe pMB1 replicon and genes which confer ampicillin and tetracyclineresistance. pBR322 is expressed at 15-20 copies per bacterial cell. pUCand pBR322 plasmids are commercially available from a number of sources(for example, Gibco BRL, Gaithersburg, Md.).

B. Regulatory Elements

i) Enhancer/Promoters

The transcription of each cDNA is directed by genetic elements whichallow for high levels of transcription in the host cell. Each cDNA isunder the transcriptional control of a promoter and/or enhancer.Promoters and enhancers are short arrays of DNA which direct thetranscription of a linked gene. While not intending to limit theinvention to the use of any particular promoters and/or enhancerelements, the following are preferred promoter/enhancer elements as theydirect high levels of expression of operably linked genes in a widevariety of cell types. The SV40 and SRα enhancer/promoters areparticularly preferred when the vector is to be transfected into a hostcell which expresses the SV40 T antigen as these enhancer/promotersequences contain the SV40 origin of replication.

a) The SV40 enhancer/promoter is very active in a wide variety of celltypes from many mammalian species [Dijkema, R. et al., EMBO J., 4:761(1985)].

b) The SRα enhancer/promoter comprises the R-U5 sequences from the LTRof the human T-cell leukemia virus-1 (HTLV-1) and sequences from theSV40 enhancer/promoter [Takebe, Y. et al., Mol. Cell. Biol., 8:466(1988)]. The HTLV-1 sequences are placed immediately downstream of theSV40 early promoter. These HTLV-1 sequences are located downstream ofthe transcriptional start site and are present as 5' nontranslatedregions on the RNA transcript. The addition of the HTLV-1 sequencesincreases expression from the SV40 enhancer/promoter.

c) The human cytomegalovirus (CMV) major immediate early gene (IE)enhancer/promoter is active in a broad range of cell types [Boshart etal., Cell 41:521 (1985)]. The 293 cell line (ATCC CRL 1573) [J. Gen.Virol., 36:59 (1977), Virology 77:319 (1977) and Virology 86:10 (1978)],an adenovirus transformed human embryonic kidney cell line, isparticularly advantageous as a host cell line for vectors containing theCMV enhancer/promoter as the adenovirus IE gene products increase thelevel of transcription from the CMV enhancer/promoter.

d) The enhancer/promoter from the LTR of the Moloney leukemia virus is astrong promoter and is active in a broad range of cell types [Laimins etal., Proc. Natl. Acad. Sci. USA 79:6453 (1984)].

e) The enhancer/promoter from the human elongation factor 1α gene isabundantly transcribed in a very broad range of cell types [Uetsuki etal., J. Biol. Chem., 264:5791 (1989) and Mizushima and Nagata, Nuc.Acids. Res. 18:5322 (1990)].

ii) Poly A Elements

The cDNA coding region is followed by a polyadenylation (poly A)element. The preferred poly A elements of the present invention arestrong signals that result in efficient termination of transcription andpolyadenylation of the RNA transcript. A preferred heterologous poly Aelement is the SV40 poly A signal (See SEQ ID NO:3). Another preferredheterologous poly A element is the poly A signal from the humanelongation factor 1α (hEF1α) gene. (See SEQ ID NO:41). The invention isnot limited by the poly A element utilized. The inserted cDNA mayutilize its own endogenous poly A element provided that the endogenouselement is capable of efficient termination and polyadenylation.

iii) Splice Junctions

The expression vectors also contain a splice junction sequence. Splicingsignals mediate the removal of introns from the primary RNA transcriptand consist of a splice donor and acceptor site. The presence ofsplicing signals on an expression vector often results in higher levelsof expression of the recombinant transcript. A preferred splice junctionis the splice junction from the 16S RNA of SV40. Another preferredsplice junction is the splice junction from the hEF1α gene. Theinvention is not limited by the use of a particular splice junction. Thesplice donor and acceptor site from any intron-containing gene may beutilized.

C. Polylinkers

The expression vectors contain a polylinker which allows for the easyinsertion of DNA segments into the vector. A polylinker is a shortsynthetic DNA fragment which contains the recognition site for numerousrestriction endonucleases. Any desired set of restriction sites may beutilized in a polylinker. Two preferred polylinker sequences are the SD5and SD7 polylinker sequences. The SD5 polylinker is formed by the SD5A(SEQ ID NO:1) and SD5B (SEQ ID NO:2) oligonucleotides and contains therecognition sites for XbaI, NotI, SfiI, SacII and EcoRI. The SD7polylinker is formed by the SD7A (SEQ ID NO:4) and SD7B (SEQ ID NO:5)oligonucleotides and contains the following restriction sites: XbaI,EcoRI, MluI, StuI, SacII, SfiI, NotI, BssHII and SphI. The polylinkersequence is located downstream of the enhancer/promoter and splicejunction sequences and upstream of the poly A sequence. Insertion of acDNA or other coding region (i.e., a gene of interest) into thepolylinker allows for the transcription of the inserted coding regionfrom the enhancer/promoter and the polyadenylation of the resulting RNAtranscript.

D. Constructs

The above elements may be arranged in numerous combinations andconfigurations to create the expression vectors of the invention. Thegenetic elements are manipulated using standard techniques of molecularbiology known to those skilled in the art [Sambrook, J. et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, New York (1989)]. Once a suitable recombinant DNAvector has been constructed, the vector is introduced into the desiredhost cell. DNA molecules are transfected into procaryotic hosts usingstandard protocols. Briefly the host cells are made competent bytreatment with calcium chloride solutions (competent bacteria cells arecommercially available and are easily made in the laboratory). Thistreatment permits the uptake of DNA by the bacterial cell. Another meansof introducing DNA into bacterial cells is electroporation in which anelectrical pulse is used to permit the uptake of DNA by bacterial cells.

Following the introduction of DNA into a host cell, selective pressuremay be applied to isolate those cells which have taken up the DNA.Procaryotic vectors (plasmids) will contain an antibiotic-resistancegene, such as ampicillin, kanamycin or tetracycline resistance genes.The preferred pUC plasmids contain the ampicillin resistance gene.Growth in the presence of the appropriate antibiotic indicates thepresence of the vector DNA.

For analysis to confirm correct sequences in the plasmids constructed,the ligation mixture may be used to transform suitable strains of E.coli. Examples of commonly used E. coli strains include the HB101 strain(Gibco BRL), TG1 and TG2 (derivatives of the JM101 strain), DH10B strain(Gibco BRL) or K12 strain 294 (ATCC No. 31446). Plasmids from thetransformants are prepared, analyzed by digestion with restrictionendonucleases and/or sequenced by the method of Messing et al., Nuc.Acids Res., 9:309 (1981).

Plasmid DNA is purified from bacterial lysates by chromatography onQiagen Plasmid Kit columns (Qiagen, Chatsworth, Calif.) according to themanufacturer's directions for large scale preparation.

Small scale preparation (i.e., minipreps) of plasmid DNA is performed byalkaline lysis [Birnboim, H. C. and Doly, J., Nuc. Acids. Res., 7:1513(1979)]. Briefly, bacteria harboring a plasmid is grown in the presenceof the appropriate antibiotic (for pUC-based plasmids ampicillin is usedat 60 μg/ml) overnight at 37° C. with shaking. 1.5 ml of the overnightculture is transferred to a 1.5 ml microcentrifuge tube. The bacteriaare pelleted by centrifugation at 12,000 g for 30 seconds in amicrocentrifuge. The supernatant is removed by aspiration. The bacterialpellet is resuspended in 100 μl of ice-cold Solution I (50 mM glucose,25 mM Tris-HCl, pH 8.0 and 10 mM EDTA, pH 8.0). Two hundred μl ofSolution II (0.2 N NaOH and 1% SDS) is added and the tube is inverted tomix the contents. 150 μl of ice-cold Solution III (3M sodium acetateadjusted to pH 4.8 with glacial acetic acid) is added and the tube isvortexed to mix the contents. The tube is then placed on ice for 3 to 5minutes. The tube is then centrifuged at 12,000 g for 5 minutes in amicrocentrifuge and the supernatant is transferred to a fresh tube. Theplasmid DNA is precipitated using 2 volumes of ethanol at roomtemperature and incubating 2 minutes at room temperature (approximately25° C.). The DNA is pelleted by centrifugation at 12,000 g for 5 minutesin a microcentrifuge. The supernatant is removed by aspiration and theDNA pellet is resuspended in a suitable buffer such as TE buffer (10 mMTris-HCl, pH 7.6, 1 mM EDTA, pH 8.0).

Expression vector DNA purified by either chromatography on Qiagencolumns or by the alkaline lysis miniprep method is suitable for use intransfection experiments.

III. Amplification Vectors

A vector encoding a structural gene which permits the selection of cellscontaining multiple or "amplified" copies of the vector encoding thestructural gene is referred to as an amplification vector. Theamplifiable gene is capable of responding either to an inhibitor or lackof an essential metabolite by amplification to increase the expressionproduct (i.e., the expression of the protein encoded by the amplifiablegene). The amplifiable gene may be characterized as being able tocomplement an auxotrophic host. For example, the gene encoding DHFR maybe used as the amplifiable marker in conjunction with cells lacking theability to express a functional DHFR enzyme. However, it is notnecessary to use an auxotrophic host cell. In a preferred embodiment thehost cell is not auxotrophic with respect to the amplifiable marker.

The invention is not limited by the use of a particular amplifiablegene. Various genes may be employed, such as the gene expressing DHFR,the CAD gene, genes expressing metallothioneins, the gene expressingasparagine synthetase, the gene expressing glutamine synthetase andgenes expressing surface membrane proteins which offer drug resistance.By blocking a metabolic process in the cells with enzyme inhibitors,such as methotrexate, for DHFR or cytotoxic agents such as metals, withthe metallothionein genes, or by maintaining a low or zero concentrationof an essential metabolite, the cellular response will be amplificationof the particular gene and flanking sequences [Kaufman and Sharp (1982)J. Mol. Biol. 159:601]. Because the process of gene amplificationresults in the amplification of the amplifiable marker and surroundingDNA sequences, it is possible to co-amplify gene sequences other thanthose encoding the amplifiable marker [Latt, et al. (1985) Mol. Cell.Biol. 5:1750]. The amplification of sequences encoding the gene ofinterest is accomplished by co-introducing sequences encoding the geneof interest and the amplifiable marker into the same host cell.

The gene encoding the protein of interest may be physically linked tothe amplifiable marker by placing both coding regions with appropriateregulatory signals on a single vector. However it is not necessary thatboth coding regions be physically located on the same vector. Becausesmall vector molecules are easier to manipulate and give higher yieldswhen grown in bacterial hosts, it is preferred that the gene of interestand the amplifiable marker gene be located on two separate plasmidvectors. Whether the amplifiable marker and the gene of interest areencoded on the same or separate vector plasmids, the vector moleculesare linearized by digestion with a restriction enzyme prior tointroduction of the vector DNAs into the host cell. The restrictionenzyme utilized is selected for its ability to cut within the plasmidbackbone of the vector but not cut within the regulatory signals or thecoding region of the amplifiable marker or gene of interest.

The amplification vector is constructed by placing the desiredstructural gene encoding the amplifiable marker into an expressionvector such that the regulatory elements present on the expressionvector direct the expression of the product of the amplifiable gene. Theinvention is illustrated by the use of a structural gene encoding DHFRas the amplifiable marker. The DHFR coding sequences are placed in thepolylinker region of the expression vector pSSD7 such that the DHFRcoding region is under the transcriptional control of the SV40enhancer/promoter. The invention is not limited by the selection of anyparticular vector for the construction of the amplification vector. Anysuitable expression vector may be utilized. Particularly preferredexpression vectors include pSSD5, pSSD7, pSRαSD5, pSRαSD7, pMSD5 andpMSD7. These expression vectors utilize regulatory signals which permithigh level expression of inserted genes in a wide variety of cell types.

IV. Selection Vectors

An expression vector encoding a selectable marker gene is referred to asa selection vector. The selectable marker may be a dominant selectablemarker. Examples of dominant selectable markers include the neo gene,the hyg gene and the gpt gene. The selectable marker may require the useof a host cell which lacks the ability to express the product encoded bythe selectable marker. Examples of such non-dominant markers include thetk gene, the CAD gene and the hprt gene.

The invention is not limited to the use of a particular selectablemarker or to the use of any selectable marker (besides the amplifiablemarker) at all. In a preferred embodiment, the host cell used is aBPRT-deficient cell line and the amplifiable marker used is DHFR.

When an HPRT-deficient cell line is utilized and this cell line producesa functional DHFR enzyme, a selectable marker encoding the HPRT enzymemay be utilized. The host cell is co-transfected with plasmidscontaining a selectable marker (HPRT), an amplifiable marker (DHFR) andone or more proteins of interest. The transfected cells are then firstselected for the ability to grow in HxAz medium (hypoxanthine andazaserine) which requires the expression of HPRT by the cell. Cellswhich have the ability to grow in HxAz medium have incorporated at leastthe selection vector encoding HPRT. Because the vector DNAs arelinearized and then introduced into the host cell by electroporation(discussed below), cells which have taken up the HPRT vector are alsolikely to have taken up the vectors encoding DHFR and the protein ofinterest. This is because the linearized vectors form long concatemersor tandem arrays which integrate with a very high frequency into thehost chromosomal DNA as a single unit [Toneguzzo, et al. (1988) Nucl.Acid Res. 16:5515].

The ability to select for transfected cells expressing HPRT facilitatesthe use of DHFR as the amplifiable marker in a cell line which is notDHFR-deficient. The use of the selectable marker allows thecircumvention of the problem of amplification of the host cell'sendogenous DHFR gene [Walls, J. D. et al., (1989), supra]. However, asdiscussed below, the present invention can be practiced without using aselectable marker in addition to the amplification vector when celllines which are not DHFR-deficient are employed.

The invention may be practiced such that no selectable marker is used.When the amplifiable marker is a dominant amplifiable marker such as theglutamine synthetase gene or where the host cell line lacks the abilityto express the amplifiable marker (such as a DHFR⁻ cell line) noselectable marker need be employed.

V. Cell Lines and Cell Culture

A variety of mammalian cell lines may be employed for the expression ofrecombinant proteins according to the methods of the present invention.Exemplary cell lines include CHO cell lines [e.g., CHO-K1 cells (ATCCCCl 61; ATCC CRL 9618) and derivations thereof such as DHFR⁻ CHO-KI celllines (e.g., CHO/dhFr-; ATCC CRL 9096), mouse L cells and BW5147 cellsand variants thereof such as BW5147.3 (ATCC TIB 47) and BW5147.G.1.4cells (ATCC TIB 48). The cell line employed may grow attached to atissue culture vessel (i.e, attachment-dependent) or may grow insuspension (i.e., attachment-independent).

BW5147.G.1.4 cells are particularly preferred for the practice of thepresent invention. BW5147.G.1.4 cells have a very rapid doubling time[i.e., a doubling time of about 12 hours when grown in RPMI 1640 mediumcontaining 10% Fetal Clone I (Hyclone)]. The doubling time or generationtime refers to the amount of time required for a cell line to increasethe number of cells present in the culture by a factor of two. Incontrast, the CHO-K1 cell line (from which the presently available dhfr-CHO-KI cell lines were derived) have a doubling time of about 21 hourswhen the cells were grown in either DMEM containing 10% Fetal Clone II(Hyclone) or Ham's F-12 medium containing 10% Fetal Clone II.

A rapid doubling time is advantageous as the more rapidly a cell linedoubles, the more rapidly amplified variants of the cell line willappear and produce colonies when grown in medium which requires theexpression of the amplifiable marker. Small differences (i.e., 1-2hours) in the doubling times between cell lines can translate into largedifference in the amount of time required to select for a cell linehaving useful levels of amplification which result in a high level ofexpression of the non-selectable gene product. The speed with which ahigh expressing cell line can be isolated may be critical in certainsituations. For example, the production of proteins to be used inclinical applications (e.g., the production of tumor-related proteins tobe used to immunize a cancer patient) requires that the protein ofinterest be expressed in useable quantities as quickly as possible sothat maximum benefit to the patient is realized.

In addition, BW5147.G.1.4 cells permit the amplification of thenon-selectable gene (which encodes the protein of interest) at a veryhigh frequency. Using the methods of the present invention, about 80% ofBW5147.G.1.4 cells which survive growth in the selective medium (e.g.,HxAz medium) will amplify the input DNA which contains the amplifiablemarker and the DNA encoding the protein of interest (as measure by theability of the cells to survive in medium containing MTX and theproduction of increased amounts of the protein of interest). That is 80%of the cells which survive growth in the selective medium will survivegrowth in medium which requires the expression of the amplifiablemarker. When cells are subjected to growth in medium containing acompound(s) which requires expression of the amplifiable marker (e.g.,growth in the presence of MTX requires the expression of DHFR), thecells which survive are said to have been subjected to a round ofamplification. Following the initial or first round of amplification,the cells are placed in medium containing an increased concentration ofthe compounds which require expression of the amplifiable marker and thecells which survive growth in this increased concentration are said tohave survived a second round of amplification. Another round ofselection in medium containing yet a further increase in theconcentration of the compounds which require expression of theamplifiable marker is referred to as the third round of amplification.

Of those transfected BW5147.G.1.4 clones which amplify in the firstround of amplification (as measured by both the ability to grow inincreased concentrations of MTX and an increased production of theprotein of interest), about 2/3 also coordinately amplify theamplifiable gene as well as the gene encoding the protein of interest inthe second round of amplification. All clones which coordinatelyamplified the amplifiable marker and the gene encoding the protein ofinterest in the second round of amplification have been found tocoordinately amplify both genes in all subsequent rounds ofamplification.

An additional advantage of using BW5147.G.1.4 cells is the fact thatthese cells are very hardy. A cell line is said to be hardy when it isfound to be able to grow well under a variety of culture conditions andwhen it can withstand a certain amount of mal-treatment (i.e., theability to be revived after being allowed to remain in medium which hasexhausted the buffering capacity or which has exhausted certainnutrients). Hardiness denotes that the cell line is easy to work withand it grows robustly. Those skilled in the art of tissue culture knowreadily that certain cell lines are more hardy than others; BW5147.G.1.4cells are particularly hardy cells.

BW5147.G.1.4 cells may be maintained by growth in DMEM containing 10%FBS or RPMI 1640 medium containing 10% Fetal Clone I. CHO-K1 cells (ATCCCCl 61, ATCC CRL 9618) may be maintained in DMEM containing 10% FetalClone II (Hyclone), Ham's F12 medium containing 10% Fetal Clone II orHam's F12 medium containing 10% FBS and CHO/dhFr- cells (CRL 9096) maybe maintained in Iscove's modified Dulbecco's medium containing 0.1 mMhypoxanthine, 0.01 mM thymidine and 10% FBS. These cell lines are grownin a humidified atmosphere containing 5% CO₂ at a temperature of 37° C.

The invention is not limited by the choice of a particular host cellline. Any cell line can be employed in the methods of the presentinvention. Cell lines which have a rapid rate of growth or a lowdoubling time (i.e., a doubling time of 15 hours or less) and which iscapable of amplifying the amplifiable marker at a reasonable ratewithout amplification of the endogenous locus at a similar or higherrate are preferred. Cell lines which have the ability to amplify theamplifiable marker at a rate which is greater than the rate at which theendogenous locus is amplified are identified by finding that the abilityof the cell to grow in increasing concentrations of the inhibitor (i.e.,the compound which requires the cell to express the amplifiable markerin order to survive) correlates with an increase in the copy number ofthe amplifiable marker (this may be measured directly by demonstratingan increase in the copy number of the ampliflable marker by Southernblotting or indirectly by demonstrating an increase in the amount ofmRNA produced from the ampliflable marker by Northern blotting).

VI. Co-Transfection of Cell Lines

Prior to introduction of vector DNA into a given cell line, the vectorDNA is linearized using a restriction enzyme which cuts once within thevector sequences and which does not cut within the control or codingregions necessary for the expression of the encoded protein.Linearization of the DNA is advantageous as it promotes the integrationof the vector DNA into the chromosomal DNA of the host cell line (freeends of DNA are recombinogenic). Furthermore, vector DNA must break inorder to integrate into the genomic DNA of the host cell; linearizationallows control over where this break occurs thereby preventing the lossof functional vector sequences by directing this break to anon-essential region of the vector. Additionally, linear DNA moleculestend to integrate into the genomic DNA of the host cell as a random headto tail concatemer (it is noted that circular DNA also tends tointegrate as a head to tail concatemer; however, as discussed above, thecircular DNA must break prior to integration). This obviates the need toconstruct a single large vector containing the selectable gene,amplifiable gene and the gene(s) of interest. Several smaller vectorsmay be co-transfected instead thereby essentially eliminating thelikelihood that the vector will suffer a break in an essential region.

To generate a stable cell line expressing large quantities of a desiredprotein(s), the following vectors are introduced as linear DNA: 1) aselectable vector such as pMSD5-HPRT; 2) an amplifiable vector such aspSSD7-DHFR and 3) one or more vectors encoding a gene of interest. Thisalso results in a much higher ratio of copies of the expressed gene(s)of interest to amplifiable marker genes in the concatemer. The ratio ofthe selectable vector, amplifiable vector and the vector(s) encoding aprotein(s) of interest is 1:2:20-50. Multiple vectors encoding separateproteins of interest are utilized when it is desirable to expressmultiple proteins in a single cell. This will be the case where theprotein of interest is a multi-chain protein. For example,immunoglobulins are formed by the association of two heavy chains andtwo light chains; the heavy and light chains are encoded by separategenes. Expression of a functional immunoglobulin requires that thetransfected cell express both the heavy and light chain genes. Up to sixnon-selectable/amplifiable plasmids (i.e., encoding a gene of interest)may be used to transfect a given cell line.

Large quantities of the expression vector(s) are introduced into thecells along with the amplification and selection vectors. Typically 10to 15 μg of the selectable vector (e.g., pMSD5-HPRT), 20 to 30 μg of theamplification vector (e.g., pSSD7-DHFR) and a total of 400 to 500 μgtotal of the expression vectors. For example, when two expressionvectors are to be used, 200 to 250 μg of each of the two expressionvectors (i.e., plasmid encoding a gene of interest) are used in additionto the selection and amplification vectors. The maximum amount of DNAwhich can be electroporated under the conditions used herein is about500 to 750 μg DNA (i.e., the total amount or the sum of all vectorDNAs). If 6 separate expression vectors are to be introduced into a cellin addition to the selection and amplification vectors, the followingamounts of DNA are employed: 7.5 μg of the selection vector, 15 μg ofthe amplification vector and ˜121 μg of each of the six expressionvectors [the total amount of DNA is therefore ˜750 μg perelectroporation using 2×10⁷ cells/ml in 0.5 ml of 1× HBS(EP)].

The vectors to be co-transfected into the cells are linearized usingappropriate restriction enzymes (i.e., enzymes which cut only within theplasmid backbone) in the same reaction tube. Following digestion withthe appropriate restriction enzymes, the DNA is precipitated usingethanol and resuspended in 0.5 ml of 1× HBS (EP) (20 mM HEPES, pH 7.0;0.75 mM Na₂ HPO₄ /NaH₂ PO₄, pH 7.0; 137 mM NaCl; 5 mM KCl and 1 gm/literdextrose).

The linearized vector DNAs are preferentially introduced into the hostcell by electroporation. Alternatively, the linearized vector DNAs maybe introduced into the host cell by microinjection using techniquesknown to the art. The use of electroporation is preferred over othermethods of introducing DNA into cells for a number of reasons: 1)efficiency of transfection. A number of attractive cell lines (e.g.,virtually any lymphoid cell line) are refractory to transformation viaany other method (such as DEAE-dextran mediated transfection or calciumphosphate-DNA co-precipitation). Electroporation of these lines allowsthe ready isolation of as many independent transformants as might bereasonably required. 2) Electroporation preserves the integrity of thetransfected DNA. DNA introduced by other methods (DEAE-dextran or CaPO₄)has been shown to acquire observable mutations at observablefrequencies, posing a concern for therapeutically used proteins derivedfrom these sorts of transfections [See for example, M. P. Calos et al.(1983) Proc. Natl. Acad. Sci. USA 80:3015; Kopchick and Stacey (1984)Mol. Cell. Biol. 4:240; Wake et al. (1984) Mol. Cell. Biol. 4:387; andLebkowski et al. (1984) Mol. Cell. Biol. 4:1951]. Lebkowski et al.,supra reported a mutation frequency in DNA chemically introduced thatwas four orders of magnitude above the endogenous mutational frequency.In contrast, DNA introduced into cells via electroporation was found tohave a mutation frequency equal to the background mutational frequencyof the cell [Drinkwater and Klinedinst (1986) Proc. Natl. Acad. Sci. USA83:3402]. 3) Cotransformation of several unlinked DNA molecules isreadily achieved using electroporation. As demonstrated herein, aminimum of four unlinked DNAs can be cotransfected into cells byelectroporation and a high frequency of the cells expressing theselectable marker will also express all of the other genes. 4)Electroporation is simple to perform. While microinjection of DNA avoidsthe increased mutation frequency observed using chemical introduction ofDNA, microinjection of somatic cells is technically challenging andrequires the use of expensive equipment. In contrast electroporation canbe performed using fairly inexpensive equipment which may be prepared inhouse or purchased commercially.

Lymphoid cell lines have been very difficult to transfect with CaPO₄-mediated co-precipitation, although it has been achieved [Rice andBaltimore (1982) Proc. Natl. Acad. Sci. USA 79:7862 and Oi et al. (1983)Proc. Natl. Acad. Sci. USA 80:825]. In contrast, transfection ofnumerous lymphoid cell lines has been achieved by electroporation withacceptably high transformation frequencies [Potter et al. (1984) Proc.Natl. Acad. Sci. USA 81: 7161; Boggs et al. (1986) Exp. Hematol. 14:988;Toneguzzo et al. (1986) Mol. Cell. Biol. 6:703 and Toneguzzo and Keating(1986) Proc. Natl. Acad. Sci. USA 83:3496]. Oi et al., supra report atransformation frequency for BW5147 cells using CaPO₄ -mediatedco-precipitation and a gpt-expressing plasmid of 1 per 10⁷ cells.Toneguzzo et al., supra report a transformation frequency for BW5147cells using electroporation and a gpt-expressing plasmid of 3.6 per 10⁴cells (a frequency greater than 3000-fold higher than that achievedusing CaPO₄ -mediated co-precipitation).

The host cells, typically BW5147.G.1.4 cells, are washed twice inice-cold 1× HBS(EP) and resuspended at 2×10⁷ cells/ml in 0.5 ml of 1×HBS(EP). The cells are then placed in a 1 ml cuvette (#67.746, Sarstedt,Inc., Princeton, N.J.) which contains the linearized DNAs. The cuvetteis placed on ice. The electroporation is performed at 225 volts using anISCO Model 493 power supply (ISCO). The electroporation apparatus isconstructed exactly as described in Chu, G. et al., Nucl. Acids Res.15:1311 (1987). The electroporation device is set on constant voltage(225 V) at the 2× setting (i.e., both capacitors are used).Alternatively, a commercially available electroporation device may beemployed [e.g., Gene Pulser™ (BioRad, Hercules, Calif.) with theCapacitance Extender set at 960 μFD]. Following electroporation, thecells are allowed to recover by incubation on ice for 5 to 15 minutes,typically 10 minutes.

VII. Selection and Co-Amplification

The electroporated cells are then transferred to a T75 flask (Falcon)containing 30 mls of RPMI 1640 medium (Irvine Scientific) supplementedwith 10% fetal calf serum (FCS; HyClone) and 50 μg/ml gentamicin(Sigma). The cells are then incubated at 37° C. in a humidifiedatmosphere containing 5% CO₂ for 36 to 48 hours. The cells are thentransferred to 48 well plates (Costar) at 1×10⁴ to 1×10⁵ cells per wellin selective medium. The use of selective medium facilitates theidentification of cells which have taken up the transfected DNA. Cellswhich grow either in an attachment-dependent manner or anattachment-independent manner are plated in multiwell plates duringgrowth in selective medium.

A variety of selectable markers may be used including both dominantselectable markers and markers which require the use of a cell linelacking a given enzyme. For example, cell lines lacking the enzyme HPRTcan be used in conjunction with a vector expressing the hprt gene. Thetransfected cells are then grown in the presence of hypoxanthine andazaserine (HxAz medium). Examples of dominant selectable markers whichdo not require the use of enzyme-deficient cell lines include the neogene, the hyg gene and the gpt gene.

When pMSD5-HPRT is used as the selectable marker, the selective mediumcomprises RPMI 1640 medium containing 10% FCS, 100 μM hypoxanthine (Hx)(Sigma) and 2 μg/ml azaserine (Az) (Sigma). After approximately 11 days,positive wells (i.e., wells containing cells capable of growth in theselective medium) are visible and the colonies are removed to 24 wellplates. The positive colonies are picked from the 48 well plates fromabout day 11 to about 3 weeks following the addition of selectivemedium.

Positive colonies removed from the 48 well plates are placed into 24well plates (Costar) in RPMI 1640 medium containing 10% dialyzed FCS(HyClone) and 100 μM Hx. The use of dialyzed serum at this pointincreases the speed and frequency of co-amplification of the input DNAin the transfectants. Hypoxanthine is retained in the culture medium fora few passages until the azaserine is diluted to non-toxicconcentrations.

The transfected cells which survived growth in selective medium are thenchecked to see if they are expressing the genes of interest. This may bedone by any suitable assay including cell surface staining, a bioassayfor activity, ELISA or immunoprecipitation followed by polyacrylamidegel electrophoresis. For example if the gene(s) of interest encode acell surface molecule, the transfected cells are analyzed by stainingwith an antibody specific for the vector-encoded cell surface molecule.The presence of the antibody on the surface of the transfected cell isdetected by fluorescence microscopy (the specific antibody is eitherdirectly conjugated to a fluorochrome or a fluorescienated secondaryantibody is utilized). The best expressing clones are then checked todetermine their level of sensitivity to MTX. Typically 6 to 18, morepreferably 12, clones are checked.

The parental (i.e., non-transfected) BW5147.G.1.4 cells barely grow inthe presence of 10 nM MTX. By visual inspection 3 to 5 days afterplating, greater than about 98 percent of the parental BW5147.G.1.4cells are killed when 1×10⁴ cells are placed in 2 ml of mediumcontaining 20 nM MTX in the well of a 24 well plate (this level of MTXis referred to as the growth cut off for the parental BW5147.G.1.4 cellline). At 30 nM MTX, colonies of BW5147.G.1.4 cells are seen at afrequency of less than 10⁻⁷.

The transfected and selected cells ("selectants") are plated in a rangeof MTX concentrations ranging from 10 to 100 nM; the cells are plated ata density of 1 to 5×10⁴ cells per well in a 24 well plate (Costar); theselectants are plated at the same density of cells as was used todetermine the level of MTX at which>about 98% of the parental cells werekilled. This is done because MTX irreversibly binds to DHFR so that thenumber of cells present in a given volume effects the concentration ofMTX required to kill the cells; that is if a higher density of cell isused, a higher concentration of MTX will be required to kill about 98%of the cells [For example when the parental cells are plated at adensity of 1×10⁴ cells/2 ml medium in the well of a 24 well plate 20 nMMTX is sufficient to kill >98% cells in a 3 to 5 day assay. If thedensity is increased two-fold (1×10⁴ cells in ml medium), 25 nM MTX isrequired for >98% killing. If 5×10⁴ cells are placed in 2 ml of mediumin the well of a 24 well plate, 30 nM MTX is required to achieve >98%killing.]

Clones of selectants typically show growth cut offs of 30 to 60 nM MTX(that is greater than about 98% of the selectants are killed when placedin medium containing 30 to 60 nM MTX when the plates are visuallyinspected 3 to 5 days after plating in medium containing this level ofMTX). Cells from each selectant of interest which shows MTX resistanceabove the parental BW5147.G.1.4 cells (e.g., above 20 to 30 nM MTX) areplated at 10⁴ cells per well of a 48 well plate (Costar) in 0.5 ml ofRPMI 1640 containing 10% dialyzed FCS and MTX (hereinafter medium-MTX).Several concentrations of MTX are used: 20 nM, 40 nM and 60 nM aboveeach clones' upper level of MTX resistance (i.e., if the upper level ofMTX resistance is 30 nM then the following concentrations may be used:50 nM, 70 nM and 90 nM); these levels of MTX are typically 4-fold to6-fold the level of MTX required to kill greater than about 98% of theparental cells. Any selectants which are capable of growth in mediumcontaining greater than 90 nM MTX are discarded; it has been observedthat selectants which are capable of growing in such high levels of MTXtend to preferentially amplify the amplification vector at the expenseof the expression vector(s).

After 7 to 10 days, the wells are fed with 0.5 ml medium-MTX. Initialamplificants are picked between 2 to 6 weeks (typically 3 to 5 weeks)after plating in medium-MTX. The clones are then analyzed again forexpression of the gene(s) of interest using the appropriate assay (i.e.,staining with antibodies for cell surface expression, ELISA, bioassaysfor activity, immunoprecipitation and PAGE, etc.).

Typically a HPRT⁺ clone is plated at a concentration of 50 to 80 nM MTX(this represent the first round of amplification). The clone is grownfor 2 to 3 weeks and then the level of MTX is increased to 200 nM to 480nM (a 4 fold increase; this represents the second round ofamplification). After another 2 to 4 weeks, the level of MTX isincreased to 1 to 2 μM MTX (another 4 to 6 fold increase; thisrepresents the third round of amplification). Any clones which show anincreased resistance to MTX without a corresponding increase inexpression of the gene(s) of interest is discarded. Typically anydiscordance is seen on the second round of amplification; such clonesprove to be unstable and are undesirable.

The methods of the present invention allow, for the first time, theco-amplification of transfected DNA sequences in BW5147 cells. Inaddition, the methods of the present invention provide improved methodsfor the co-amplification of DNA sequences in cell lines. Of theselectants that are expressing the gene(s) of interest, most (i.e.,greater than 80%), if not all, will co-amplify the amplifiable marker(e.g, the dhfr gene which confers resistance to MTX) and the gene(s) ofinterest in the first round of amplification. More than 60% of the firstround amplificants will co-amplify the gene(s) of interest in the secondround in addition to dhfr gene sequences. To date, using the methods ofthe present invention, no clones have been obtained that amplify thegene(s) of interest in the second round of amplification that then failto continue to coordinately amplify in all subsequent rounds until amaximum expression level is reached. Thus, the methods of the presentinvention result in a much higher frequency of coordinateco-amplification of gene sequences than has been reported for othermethods of co-amplification such as that reported by Walls et al. (1989)Gene 81:139 or by Kaufman et al. (1985) Mol. Cell. Biol. 5:1750 whensingle clones were examined. In addition to providing a means forachieving a very high frequency of coordinate co-amplification of genesequences, the methods of the present invention also provide methodswhich produce the desired amplificants with a considerable time savingsrelative to existing methods. The method of the present invention avoidsthe time-consuming step of searching through pools of primarytransformants which have been subjected to a round of amplification tofind those few clones within the pool which are expressing the proteinof interest at high levels.

The following modifications to the above-described amplificationprotocol have been found to decrease the amount of time required for thefirst round of amplification by 2 to 3 weeks. First, the originaltransfectants are selected by growth in RPMI 1640 medium containing 100μM Hx, 2 μg/ml Az and 10% dialyzed FCS. Second, the originaltransfectants are fed at about 10 days following electroporation with0.5 ml per well (in a 48 well plate) of RPMI 1640 medium containing 10%dialyzed FCS, 100 μM Hx and 10 nm MTX; this yields a final concentrationin each well of the 48 well plate of 5 nM MTX. The net effect of thegrowth of the transfected cells in medium containing dialyzed FCS and 5nM MTX is to give the cells which have undergone amplification events aselective advantage.

VIII. Co-Amplification Without Prior Selection

The amplified cell lines of the present invention may be generated usingonly an amplification vector in addition to the expression vector(s)(i.e., the use of a selection vector is not required). Cell lines whichdo not lack a functional gene product corresponding to the enzymeencoded by the amplification vector and which can be successfullyemployed without the use of a selectable marker in addition to theampliflable marker are those cell lines in which the background level ofamplification of the endogenous gene (e.g., the endogenous dhf gene whenDHFR is used as the amplifiable marker) is low enough that amplificationof the input amplifiable gene (i.e., the amplification vector) occurspreferentially.

When it desired that no selection step be employed, the above protocolsare modified as follows. The amplification vector and expressionvector(s) are linearized and electroporated into the parental cell lineusing a ratio of 1:10-15 (amplification vector:expression vector). Againlarge amounts of DNA are introduced, preferably by electroporation, intothe cells. Typically, 20 μg of the amplification vector is used and 200to 250 μg each of two expression vectors (or 400 to 500 μg of a singleexpression vector). Following electroporation, the transfected cells areallowed to recover for 36 to 48 hours as described above. Thetransformed cells are then transferred to 48 well plates at a density ofno more than 1×10⁶ cells per well in medium containing 4-fold to 6-foldthe concentration of inhibitor required to prevent the growth of theparental cells. Using the BW5147.G.1.4 cell line, the expected frequencyof generating a primary transformant which contains enough copies of theinput amplifiable gene to allow the isolation of a first roundamplificant capable of growth in medium containing 4- to 6-fold thelevel of inhibitor required to prevent growth of the parentalBW5147.G.1.4 cells is approximately 1 in 10⁸ to 1 in 10¹⁰ cells.Accordingly, at least 5×10⁸ to 1×10¹¹ cells must be plated in mediumcontaining elevated levels of the inhibitor to permit the isolation ofseveral first round amplificants. Cells capable of growing in 4- to6-fold the level of inhibitor required to prevent growth of the parentalcells are examined for the ability to express the protein(s) ofinterest; those clones expressing high levels of the protein of interestare subjected to subsequent rounds of amplification as described above.Any clones which do not display a coordinate increase in the level ofexpression of the protein(s) of interest and the level of resistance tothe inhibitor at any amplification step are discarded.

The ability to generate amplified cell lines without the need to employa selection vector reduces the amount of time required to produced thedesired amplified cell line. However, the use of a selection vector andthe initial selection step is advantageous particularly when workingwith cell lines which have a high background frequency of amplificationof the endogenous locus corresponding to the amplifiable gene present onthe amplification vector. Even when working with a cell line which doesnot a have a high background level of amplification of the endogenousgene, the use of a selection vector and an initial selection step isadvantageous because it allows one to work with only the primaryselectants expressing the highest levels of the gene(s) of interest.This reduces the amount of time and effort required to generateamplified cell lines expressing very high levels of the protein(s) ofinterest.

IX. High-Level Expression of Interleukin 10 in Amplified Cell Lines

Using the methods of the present invention, cell lines were isolatedwhich express large quantities of interleukin 10 (IL-10). IL-10 is acytokine produced by TH₂ cells (type 2 helper T cells),macrophages/monocytes, and some B cells. IL-10 acts to inhibit thesynthesis of cytokines by activated TH₁ cells, activated macrophages andnatural killer cells [Mosmann (1993) Ann. Rev. Immunol. 11: 165 andMosmann (1994) Advances in Immunol. 56: 1]. Studies have shown thatIL-10 expression is positively correlated with graft outcome intransplantation [Bromberg (1995 Curr. Op. Immunol. 7:639]. Accordingly,there is interest in using IL-10 therapeutically. Therapeutic use ofIL-10, of course, requires the ability to produce large quantities ofIL-10.

Presently, there are two commercial sources of murine IL-10. GenzymeDiagnostics (Cambridge, Mass.) sells 5 mg of IL-10 produced in E. coliproduced for $295.00 (cat#2488-01, ˜2500 units). Biosource International(Camarillo, Calif.) sells 5 mg of IL-10 produced in E. coli for $245.00(cat# PMC-0104, ˜2500 units). The methods of the present invention wereused to isolate cell line which produces about 75,000 units permilliliter of culture supernatant. Using the lower commercial price forIL-10, these cells produce about $7,350,000.00 worth of IL-10 per literin a static culture. These amplified cell lines yield about 150 mg ofIL-10 protein per liter in static culture; thus, the unpurified culturesupernatant from these amplified cell lines provides a much more puresource of IL-10 than do presently available commercial preparations.

X. High-Level Expression of Human Class II MHC Antigens and T CellReceptor Proteins in Amplified Cell Lines

The human class II MHC antigens, HLA-DR, and their corresponding mouseanalogs, the Ia antigens, are cell surface membrane glycoproteins whichmediate the recognition of non-self molecules (i.e., antigens) by Tlymphocytes. Class II molecules display fragments of foreign antigens onthe surface of antigen presenting cells which include macrophages,dendritic cells, B lymphocytes and activated T lymphocytes. WhenMHC-restricted, antigen-specific T lymphocytes interact with antigenpresenting cells bearing class II molecules bound to antigen, an immuneresponse is generated.

Class II antigens comprise two chains, an a chain and a β chain. Bothchains must be expressed in the same cell in order for the class IImolecule to be transported to the surface of the cell. The β chain ishighly polymorphic and this polymorphism results in heritabledifferences in immune responsiveness. In certain class II MHC molecules(e.g., mouse IA, human DQ and DP), the α chain is also highlypolymorphic. Given the central role that class II molecules play in theimmune response, including rejection of transplanted tissue andheritable susceptibility to autoimmune disease, studies of theinteraction of class II molecules with foreign antigen and with Tlymphocytes have been undertaken. These studies of the physical-chemicalinteraction of class II molecules with antigen require the availabilityof large quantities of purified soluble class II molecules. In addition,the use of class II molecules complexed with specific peptides has beensuggested for the treatment of autoimmune disease [Sharma, et al. (1991)Proc. Natl. Acad. Sci. USA 88:11465].

In order to provide such reagents, chimeric human DR molecules wereexpressed at high levels on the surface of amplified cell lines usingthe selection amplification method of the invention. The DR moleculesare cleaved from the cell surface to produce soluble DR molecules bytreatment with an enzyme capable of cleaving either aphosphatidylinositol linkage or a thrombin site which is present on thechimeric DR molecule.

A similar approach allows the production of large quantities of solubleT cell receptor (TCR) molecules or immunoglobulin (Ig) molecules. Like,class II molecules, TCR and Ig molecules comprise heterodimers (i.e.,two different chains associate to form the TCR or Ig molecule displayedon the cell surface; it is noted that both cell surface and solubleforms of Ig molecules exist in nature and for patient immunization onewould produce soluble Ig). The methods of the present invention permitthe production of large quantities of soluble forms of class II MHCmolecules and TCR to be produced in a rapid manner. This allowing forthe production of customized tumor cell vaccines comprising soluble TCRfor the treatment of lymphoma and leukemia patients as well as theproduction of soluble class II MHC molecules for the treatment ofautoimmune disease.

XI. Production of Custom Multivalent Vaccines for the Treatment ofLymphoma and Leukemia

The existing approach toward vaccination (ie., active immunotherapy) ofB-cell lymphoma and leukemia involves the production of a custom vaccinecomprising autologous immunoglobulin idiotype which corresponds to themost abundant antibody molecule expressed on the surface of the B-celltumor. An analogous approach for the treatment of T-cell lymphomas andleukemias would involve the production of a custom vaccine comprisingautologous T cell receptor (TCR) idiotype which corresponds to the mostabundant TCR molecule expressed on the surface of the T-cell tumor.

It is known in B-cells that the variable regions of rearrangedimmunoglobulin (Ig) genes accumulate point mutations following antigenicstimulation (Ig). This process, known as somatic mutation or somatichypermutation, is responsible for affinity maturation of humoral immuneresponses [Tonegawa (1983) Nature 302:575, Teillaud et al. (1983)Science 222:721, Griffiths et al. (1984) Nature 312:272 and Clarke etal. (1985) J. Exp. Med. 161:687]. During affinity maturation, antibodiesof higher affinity emerge as the immune response proceeds (i.e.,progression from primary to secondary to tertiary responses). Acomparison of the antibody produced during the immune response revealsthat mutations accumulate from the late stage of primary responsesonward; these mutations cluster in the second complementaritydetermining region (CDR2) region of the Ig molecule (i.e., within thehypervariable regions within the antigen-binding site). Somatic mutationdoes not occur in T cells.

Somatic variants are known to exist within the population of cellscomprising certain B-cell tumors (e.g., low grade or follicular B-celllymphomas); thus, while these tumors are clonal at the level of Ig generearrangements (including nucleotide sequence at the V-D-J junctions)they are in fact quasi-clonal with respect to the nucleotide or aminoacid sequence of their heavy chain V regions [Cleary ML et al. (1986),Cell 44:97 and Levy S et al. (1988) J. Exp. Med. 168:475]. It is thoughtthat following the transformation event(s) which gives rise to theB-cell tumor, somatic mutation persists. Analysis of B-cell lymphomasreveals that about 1 to 5% of the cells within the tumor contain somaticmutations.

The fact that somatic variants exist within a B-cell tumor hasimplications for immunotherapy of these tumors. For example, treatmentof B-cell lymphoma with anti-idiotype antibodies was shown to produce aninitial partial response in patients; however, idiotype variant tumorcells (idiotype negative) emerged at the original tumor site [Cleary MLet al. (1986), supra; Bahler DW and Levy R (1992) Proc. Natl. Acad. Sci.USA 89:6770; Zelenetz AD et al. (1992) J. Exp. Med. 176:1137; and Zhu Det al. (1994) Brit. J. Haematol. 86:505]. It is thought that theseidiotype variant tumor cells were already present before treatment withthe monoclonal anti-idiotype antibody and that they were allowed toproliferate after the selective removal of the idiotype positive tumorcells. These clinical trials showed the drawback of targeting a singleepitope on the tumor cell.

In order to improve the immunotherapy of B-cell tumors, activeimmunization with autologous tumor derived Ig or Ig subfragments hasbeen employed. It is hoped that the use of the Ig or Ig subfragments asan immunogen would produce a T cell response and antibodies directedagainst a number of different epitopes found within the tumor-specificIg. In this type of treatment the Ig (or idiotype fragment of the Ig) ofthe patient's tumor cell is expressed. While this approach has theadvantage that multiple epitopes are targeted, it still suffers from thefact that a single Ig (or subfragment) is used as the immunogen andtherefore the possibility exists that tumor cells expressing somaticvariants of the predominant Ig will escape and proliferate. To producethe tumor Ig-idiotype protein used for immunization with existingmethodologies, lymphoma cells removed by surgical biopsy are fused withthe heterohybridoma cell line K6H6/B5 which has lost the ability tosecrete endogenous Ig. Hybrid cells which secrete Ig corresponding tothe immunophenotype of the tumor sample are expanded and the secreted Igis purified for use as a vaccine [Kwak et al. (1992), supra]. Thistechnique is referred to as "rescue fusion." The Ig produced by rescuefusion represents a single Ig derived from the patient's tumor; this Igis presumably the predominant Ig expressed by the tumor. Thus, vaccinesproduced by rescue fusion are monovalent and do not represent the fullcomplexity of Ig expressed by tumors which contain somatic variants.

Clinical trials using tumor Ig-idiotype protein produced by rescuefusion to vaccinate B-cell lymphoma patients are in progress. Thesetrials are showing impressive clinical improvements for these tumorswhich remain essentially incurable with conventional therapy (i.e.,chemotherapy). This custom vaccine therapy is used following a course ofconventional chemotherapy (employed to reduce the tumor burden). Theclinical improvements are seen when comparing patients treated solelywith conventional chemotherapy with patients who received customvaccines following chemotherapy. Among the patients who have beentreated with custom vaccines and followed for a lengthy period of time(about 8 years), one has recently relapsed. Although not confirmed atthis time, it is possible that this relapse is due to the outgrowth oftumor cells bearing somatic variants of the tumor Ig-idiotype proteinused in the vaccine.

In addition to the failure to provide a multivalent vaccinerepresentative of all Ig variants present in the patients tumor, therescue fusion technique has other draw backs. This technique requires alarge number of tumor cells which are obtained by surgical biopsy ofenlarged lymph nodes in the patient. Some B-cell lymphoma patients donot present with enlarged lymph nodes suitable for surgical biopsy andtherefore cannot be treated using vaccines produced by the rescue fusiontechnique. Furthermore, the production of each custom heterohybridomacell line secreting the patients Ig takes between 2 to 8 months (averageof 4 months) and is labor intensive; in laboratories having extensiveexperience with the rescue fusion technique, the rate of vaccineproduction is about 25 custom vaccines per technician per year. Thisrate of producing custom vaccines is not sufficient to meet the existingand growing patient demand.

Ideally, the method for producing custom tumor specific vaccines couldbe performed on a small number of cells (i.e., from a fine needlebiopsy), would produce a multivalent vaccine representative of the fullcomplexity of the patient's tumor, would be fast (average of 2-3 months)and would be less labor intensive than existing methods such that asingle technician could produce at least a hundred custom vaccines peryear.

The methods described herein (Examples 9 and 10) provide a means toproduce custom vaccines, including multivalent vaccines, from smallnumbers of cells quickly and efficiently. The ability to use a smallsample size permits the production of custom vaccines for patientslacking enlarged lymph nodes suitable for surgical biopsy. In additionto expanding the pool of patients who can be treated with customvaccines, the ability to use fine needle biopsy material obviates theneed for surgery for all lymphoma patients (at least with respect to thecollection of cells for the production of custom vaccines).

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: M (molar); mM (millimolar); μM (micromolar); nM(nanomolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); gm (grams); mg (milligrams); μg (micrograms);pg(picograms); L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C.(degrees Centigrade); AMP (adenosine 5'-monophosphate); cDNA (copy orcomplimentary DNA); DNA (deoxyribonucleic acid); ssDNA (single strandedDNA); dsDNA (double stranded DNA); dNTP (deoxyribonucleotidetriphosphate); RNA (ribonucleic acid); PBS (phosphate buffered saline);g (gravity); OD (optical density); HEPES(N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]); HBS (HEPESbuffered saline); SDS (sodium dodecylsulfate); Tris-HCl(tris[Hydroxymethyl]aminomethane-hydrochloride); Klenow (DNA polymeraseI large (Klenow) fragment); rpm (revolutions per minute); EGTA (ethyleneglycol-bis(β-aminoethyl ether) N, N, N', N'-tetraacetic acid); EDTA(ethylenediaminetetracetic acid); bla (β-lactamase orampicillin-resistance gene); ORI (plasmid origin of replication); laci(lac repressor); Amicon (Amicon Corp., Beverly, Mass.); ATCC (AmericanType Culture Collection, Rockville, Md.); Becton Dickinson (BectonDickinson Immunocytometry Division, San Jose Calif.); Costar (Costar,Cambridge, Mass.); Falcon (division of Becton Dickinson Labware, LincolnPark, N.J.); FMC (FMC Bioproducts, Rockland, Me.); Gibco/BRL (Gibco/BRL,Grand Island, N.Y.); HyClone (HyClone, Logan, Utah); Sigma (SigmaChemical Co., St. Louis, Mo.); NEB (New England Biolabs, Inc., Beverly,Mass.); Operon (Operon Technologies, Alameda, Calif.); Perkin-Elmer(Perkin-Elmer, Norwalk, Conn.); Pharmacia (Pharmacia Biotech,Pisacataway, N.J.); Promega (Promega Corp., Madison, Wis.); Sarstedt(Sarstedt, Newton, N.C.); Stratagene (Stratagene, LaJolla, Calif.); U.S.Biochemicals (United States Biochemical, Cleveland, Ohio.); and Vector(Vector Laboratories, Burlingame, Calif.).

EXAMPLE 1

Construction of Expression Vectors

In order to construct the expression vectors of the invention a numberof intermediate vectors were first constructed.

Construction of pSSD5 and pSSD7

pSSD5 and pSSD7 contain the following elements from SV40: theenhancer/promoter region, the 16S splice donor and acceptor and the polyA site. Vectors containing the SV40 enhancer/promoter sequences willreplicate extrachromosomally in cell lines which express the SV40 largeT antigen as the SV40 enhancer/promoter sequences contain the SV40origin of replication.

A polylinker containing the recognition sequences for severalrestriction enzymes is located between the splice acceptor and poly Asequences. The polylinker allows for the easy insertion of a gene ofinterest. The gene of interest will be under the transcriptional controlof the SV40 enhancer/promotor. pSSD5 and pSSD7 differ only in thesequences of the polylinker (sequences listed below). The polylinker ofpSSD5 contains the following restriction sites: XbaI, NotI, SfiI, SacIIand EcoRI. The polylinker of pSSD7 contains the following restrictionsites: XbaI, EcoRI, MluI, StuI, SacII, SfiI, NotI, BssHII and SphI.

pSSD5 was constructed by digestion of the plasmid pL1 [Okayama and Berg,Mol. Cell. Biol., 3:280 (1983)] with PstI and HindIII. All restrictionenzymes were obtained from New England Biolabs and were used accordingto the manufacturer's directions. The plasmid pcDV1 [Okayama and Berg,supra] was digested with HindIII and BamHI. Both digests wereelectrophoresed on a 0.8% low melting temperature agarose gel(SeaPlaque, FMC). A 535 bp DNA fragment from the pL1 digest containingthe SV40 enhancer/promoter and 16S splice junctions was cut out of thegel. A 2.57 kb DNA fragment from the pcDV1 digest containing the SV40polyadenylation signals and the pBR322 backbone was cut out of the gel.The two gel slices were combined in a microcentrifuge tube and theagarose was removed by digestion with β-Agarase I (NEB) followed byisopropanol precipitation according to the manufacturer's directions.The DNA pellets were dried and resuspended in 20 μl of TE.

Two synthetic oligonucleotides (Operon), SD5A [5'-TCTAGAGCGGCCGCGGAGGCCGAATTCG-3' (SEQ ID NO:1)] and SD5B [5'-GATCCGAATTCGGCCTCCGCGGCCGCTCTAGATGCA-3' (SEQ ID NO:2)] were added in equal molar amountsto the resuspended DNA fragments. Ligation buffer (10× concentrate, NEB)was added to a 1× concentration, 80 units of T4 DNA ligase was added andthe ligation was placed at 14° C. overnight. Competent E. coli cellswere transformed with the ligation mixture and a plasmid was isolatedthat consisted of the DNA fragments from pL1 and pcDV1 with a novelpolylinker connecting the fragments. The resulting plasmid was namedpSSD.

The ˜670 bp BamHI/PstI fragment containing the SV40 poly A sequences(SV40 map units 2533 to 3204; SEQ ID NO:3) was removed from SV40 DNA andcloned into pUC19 digested with BamHI and PstI. The resulting plasmidwas then digested with BclI (corresponds to SV40 map unit 2770). Theends were treated with the Klenow enzyme (NEB) and dNTPs to create bluntends. Unphosphorylated PvuII linkers (NEB) were ligated to the bluntedends and the plasmid was circularized to create pUCSSD. The SV40 poly Asequences can be removed from pUCSSD as a BamHI/PvuII fragment.

pSSD5 was constructed by ligating together the following threefragments: 1) the 1873 bp SspI/PvuII fragment from pUC19; this providesthe plasmid backbone; 2) the 562 bp fragment containing the SV40enhancer/promoter and 16S splice junction and the polylinker from PSSD;this fragment was obtained by digestion of pSSD with SspI and partialdigestion with BamHI followed by isolation on low melting agarose andrecovery as described above; and 3) the 245 bp BamHI/PvuII fragment frompUCSSD (this fragment contains the SV40 poly A sequences). The threefragments were mixed together and ligated using T4 DNA ligase (NEB) tocreate pSSD5. The map of pSSD5 is shown in FIG. 1.

To create pSSD7, pSSD5 was digested with XbaI and EcoRI. The syntheticoligonucleotide pair SD7A and SD7B (Operon) was ligated into the cutpSSD5 thereby generating the SD7 polylinker. The sequence of SD7A is5'-CTAGAATTC ACGCGTAGGCCTCCGCGGCCGCGCGCATGC-3' (SEQ ID NO:4). Thesequence of SD7B is 5'-AATTGCATGCGCGCGGCCGCGGAGGCCTACGCGTGA ATT-3' (SEQID NO:5). The map of pSSD7 is shown in FIG. 2.

Construction of pSRαSD5 and pSRαSD7

pSRαSD5 and pSRαSD7 contain the SRα enhancer/promoter followed by the16S splice junction of SV40 and either the polylinker formed by theoligonucleotide pair SD5A/SD5B or SD7A/SD7B. The polylinker is followedby the SV40 poly A sequences. A gene of interest can be inserted intothe polylinker and the expression of the inserted gene will be under thecontrol of the human SRα enhancer/promoter. The SRα enhancer/promoters ahybrid enhancer/promoter comprising human T cell leukemia virus 1 5'untranslated sequences and the SV40 enhancer [Takebe et al., Mol. Cell.Biol., 8:466 (1988)]. The SRa enhancer/promoter is reported to increaseexpression from the SV40 enhancer/promoter by ten-fold in host cells.This enhancer/promoter is active in a broad range of cell types. Vectorscontaining the SRα enhancer/promoter will replicate in cells expressingSV40 large T antigen as the SV40 origin of replication is present withinthe SRa enhancer/promoter sequences.

The SRα enhancer/promoter was removed from pcDL-SRα296 by digestion withHindIII and XhoI. The ˜640 bp HindIII/XhoI fragment (SEQ ID NO:6) wasrecovered from a low melting agarose gel as described above. This ˜640bp fragment was inserted into either pSSD5 or pSSD7 digested withHindIII and XhoI (removes the SV40 enhancer/promoter from pSSD5 orpSSD7). The map of pSRαSD5 is shown in FIG. 3. The map of pSRαSD7 isshown in FIG. 4.

Construction of pMSD5 and pMSD7

pMSD5 and pMSD7 contain the long terminal repeat (LTR) from the Moloneymurine leukemia virus followed by the 16S splice junction of SV40 andeither the polylinker formed by the oligonucleotide pair SD5A/SD5B orSD7A/SD7B. The polylinker is followed by the SV40 poly A sequences. Agene can be inserted into the polylinker and the expression of theinserted gene will be under the control of the Moloney LTR.

The Moloney LTR was removed from a plasmid containing Moloney murineleukemia viral DNA [Shinnick et al., Nature 293:543 (1981)] by digestionof the plasmid with ClaI (corresponds to Moloney map unit 7674). Theends were made blunt by incubation with Klenow and dNTPs.Unphosphorylated HindIII linkers (NEB) were ligated onto the blunt ends.This treatment destroyed the ClaI site and replaced it with a HindIIIsite. The plasmid was then digested with SmaI (corresponds to Moloneymap unit 8292) and unphosphorylated XhoI linkers were ligated onto theends. The resulting plasmid now contains a XhoI site replacing the SmaIsite at Moloney map unit 8292 and a HindIII site replacing the ClaI siteat Moloney map unit 7674. The plasmid was then digested with XhoI andHindIII. The resulting XhoI/HindIII fragment containing the Moloney LTR(SEQ ID NO:7) was inserted into pSSD5 digested with XhoI and HindIII(this removes the SV40 enhancer/promoter and 16S splice junction frompSSD5) to yield pMSD5. The map of pMSD5 is shown in FIG. 5.

To create pMSD7, the Moloney LTR on the XhoI/HindIII fragment wasinserted into pSSD7 digested with XhoI and HindIII. The map of pMSD7 isshown in FIG. 6.

Construction of Vectors Containing the Human Elongation Factor 1αEnhancer/Promoter

The human elongation factor 1α enhancer/promoter is abundantlytranscribed in a very broad range of cell types. Vectors containing twoversions of this active enhancer/promoter were constructed: 1) a longversion containing ˜1.45 kb of sequences located upstream of theinitiation codon and continuing through the first intron to provide asplice junction and 2) a short version containing 475 bp of sequencesupstream of the initiation codon. The short version of the promoter istermed the "A" version and the long version is termed the "B" version.

A. Construction of pHEF1αSD5 and pHEF1αASD7

pHEF1αASD5 and pHEF1αASD7 contain the short version of the humanelongation factor 1α enhancer/promoter [Uetsuki et al., J. Biol. Chem.,264:5791 (1989) and Mizusbima and Nagata, Nuc. Acids. Res., 18:5322(1990)]. The human elongation factor 1α enhancer/promoter is abundantlytranscribed in a very broad range of cell types including L929, HeLa,CHU-2 and COS cells.

The human elongation factor 1α enhancer/promoter (nucleotides 125 to 600of the human elongation factor 1α gene; SEQ ID NO:8) was isolated fromhuman genomic DNA as follows. Genomic DNA was isolated from the MOU cellline (GM 08605, NIGMS Human Genetic Mutant Cell Repository, Camden,N.J.) using standard techniques [Sambrook et al., supra at pp.9.16-9.23]. The MOU cell line is an Epstein-Barr virus transformed humanB cell line.

Two synthetic oligonucleotide primers (Operon) were used to prime thepolymerase chain reaction (PCR) for the isolation of an ˜475 bp fragmentcontaining the human elongation factor 1α enhancer/promoter (SEQ IDNO:8). U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,965,188 cover PCRmethodology and are incorporated herein by reference.

The 5' primer, designated HBEF1αL5, contains the following sequence:5'-AAGCTTTGGAGCTAAGCCAGCAAT-3' (SEQ ID NO:9). The 3' primer, designatedHEF1αL3A, contains the following sequence: 5'-CTCGAGGCGGCAAACCCGTTGCG-3' (SEQ ID NO:10). PCR conditions were as reported in Saikiet al., Science 239:487 (1988). Briefly, 10 μg MOU genomic DNA and 1 μMfinal concentration of each primer were used in a 400 μl PCR reaction.Reaction conditions were 94° C. for 1 minute, 60° C. for 1 minute, 72°C. for 1.5 minutes, 30 cycles. Taq DNA polymerase was obtained fromPerkin-Elmer. The primer pair generates a 475 bp fragment having aHindIII site at the 5' end and a XhoI site at the 3' end. The PCRreaction products were electrophoresed on a low melting agarose gel andthe 475 bp fragment was recovered as described above. The recoveredfragment was digested with HindIII and XhoI and inserted into eitherpSSD5 or pSSD7 digested with HindIII and XhoI to yield pHEF1αASD5 andpHEF1αASD7, respectively. The maps of pHEF1αASD5 and pHEF1αASD7 areshown in FIG. 7 and 8, respectively.

B. Construction of pHEF1αBSD5 and pHEF1αBSD7

pHEF1αBSD5 and pHEF1αBSD7 were constructed as described above forpHEF1αASD5 and pHEF1αASD7 with the exception that the HEF1αL3B primerwas used instead of the HEF1αL3A primer with the HEF1αL5 primer togenerate a ˜1.45 kb fragment containing the human elongation factor 1αenhancer/promoter and a splice donor and acceptor from the humanelongation factor 1α gene. The ˜1.45 kb fragment corresponds to mapunits 125 to 1567 in the human elongation factor 1α gene (SEQ ID NO:11).The sequence of HBEF1αL3B is 5'-TCTAGAGTTTTCACG ACACCTGA-3' (SEQ IDNO:12). The HEF1αL3B primer generates a XbaI site at the 3' end of the˜1.45 kb fragment. This fragment was digested with HindIII and XbaI andinserted into either pSSD5 or pSSD7 digested with HindIII and XbaI togenerate pHBEF1αBSD5 or pHEF1αBSD7, respectively. Digestion of pSSD5 andpSSD7 with HindIII and XbaI removes the SV40 enhancer/promoter and theSV40 16S splice junction. These SV40 sequences are replaced with thehuman elongation factor 1α enhancer/promoter and a splice donor andacceptor from the human elongation factor 1α gene. The maps ofpHEF1αBSD5 and pHEF1αBSD7 are shown in FIGS. 9 and 10, respectively.

EXAMPLE 2 Construction of the Selection Vector pMSD5-HPRT

pMSD5-HPRT contains a full length cDNA clone encoding the mouse BPRTenzyme under the transcriptional control of the Moloney LTR. The MoloneyLTR contains a strong enhancer/promoter which is active in a broad rangeof cell types [Laimins et al., Proc. Natl. Acad. Sci. USA 79:6453(1984)]. The pMSD5-HPRT expression vector is used as the selectiveplasmid (or selective or selectable marker) when HPRT- cell lines, suchas BW5147.G.1.A, are used as the recipient cell line for the generationof stable transformants. HPRT⁻ cell lines cannot grow in mediumcontaining hypoxanthine, aminopterin or azaserine and thymidine (HATmedium). The addition of a functional HPRT gene by gene transfer allowsthe cells which have integrated the vector DNA encoding the HPRT gene togrow in HAT medium.

a. Isolation of a Full Length Mouse HPRT cDNA

A cDNA library was prepared from poly A⁺ mRNA isolated from C6VL cells[Allison et al., J. Immunol., 129:2293 (1982)] using standard techniques[Sambrook et al., supra at 7.26-7.29]. cDNA was generated from the mRNAand inserted into the expression vector λgt10 using standard techniques[Huynh, et aL, in DNA Cloning: A Practical Approach (D. M. Glover, ed.),Vol. 1, IRL Press Oxford (1985), pp. 49-78]. The full-length mouse HPRTcDNA was isolated using a full-length human HPRT cDNA clone containingan approximately 1.4 kb PstI-BamHI restriction fragment as a probe[pcD-HPRT; Jolly et al. (1983) Proc. Natl. Acad. Sci. USA 80:477]. Thefull length cDNA clone was digested with NotI and EcoRI to generate a1.3 kb fragment containing the coding region of HPRT (the coding regionof the mouse HPRT is listed in SEQ ID NO:13; the amino acid sequenceencoded within SEQ ID NO:13 is listed in SEQ ID NO:14).

pMSD5 (described in Example 1) was digested with NotI and EcoRI and the1.3 kb NotI/EcoRI fragment containing the mouse HPRT cDNA was insertedto generate pMSD5-HPRT. The map of pMSD5-HPRT is shown in FIG. 11.

EXAMPLE 3 Construction of the Amplification Vector pSSD7-DHFR

pSSD7-DHFR contains a full length copy of the mouse DHFR cDNA under thetranscriptional control of the SV40 enhancer/promoter. Thispromoter/enhancer is active in a wide variety of cell types from manymammalian species [Dijkema et al., EMBO J., 4:761 (1985)]. pSSD7-DHFR isreferred to as the ampliflable marker as the use of this vector allowsthe selection of cell lines which have amplified the vector sequences byselecting for cell which can grow in increasing concentrations of MTX.

The mouse DHFR cDNA was isolated from double stranded cDNA generatedfrom liver RNA using the PCR as follows. Poly A⁺ RNA was isolated fromthe liver of (Balb/c × C57B1/6) F1 mice using standard techniques. Firststrand cDNA was synthesized from the poly A⁺ RNA in a final reactionvolume of 100 μl. The following reagents were added in order: 35.6 μl H₂O, 5 μl poly A⁺ RNA (1 μg) and 1.4 μl SBNSSdT primer (1 μg). Thesequence of the SBNSSdT primer is 5'-GCATGCGCGCGGCCGCGGAGGCTTTTTTTTTTTTTTTTTT-3' (SEQ ID NO: 15). The water,primer and RNA were heated at 60° C. for 2 minutes then placed on ice.Forty μl of all four dNTPs at 5 mM each, 10 μl 10× reverse transcriptasesalts (1.0 M Tris-HCl, pH 8.3, 0.5 M KCl, 0.1 M MgCl₂, 0.1 M DTT), 2 μlRNasin (Promega) and 5 μl AMV reverse transcriptase (Molecular GeneticResources, Tampa, Fla.). The reaction was run at 41° C. for 3 hours. Thereaction was stopped by incubation at 65° C. for 10 minutes.

The reaction components were transferred to a Centricon 100 tube(Amicon) and 2.1 ml of 5 mM Tris-HC1, pH 8.3 was added. The tube wascentrifuged at 300 rpm (˜700 g) for 4 minutes at 10° C. 2.2 ml ofTris-HCl, pH 8.3 was added and the tube was centrifuged again as above.This washing step was repeated and then the tube was inverted andcentrifuged at 2500 rpm for 5 minutes at 10° C. to recover the firststrand cDNA (volume ˜50 μl). Second strand cDNA was synthesized asfollows. 96 μl H₂ O and 20 μl 10× rTth RTase buffer (900 mM KCl, 100 mMTris-HCl, pH 8.3) was added to the first strand cDNA. In a separate tubethe following components were mixed: 20 μl 10 mM MnCl₂, 4 μl of each ofthe four dNTPs at 10 mM and 10 μl rTth reverse transcriptase(Perkin-Elmer). Both mixtures were heated to 60° C. and the secondmixture was added to the cDNA mixture. The reaction was carried out at60° C. for 10 minutes. The reaction was stopped by addition of 25 μlchelating buffer [50% glycerol (v/v), 1 mM KCl, 100 mM Tris-HCl, pH 8.3,7.5 mM EGTA, 0.5% Tween 20] and the mixture was placed on ice.

The reaction mixture was then transferred to a Centricon 100 tube and2.1 ml of 5 mM Tris-HCl, pH 7.5 was added. The tube was centrifuged at5500 rpm for 30 minutes at 10° C. 2.2 ml of Tris-HCl, pH 7.5 was addedand the tube was centrifuged again as above. This washing step wasrepeated and then the tube was inverted and centrifuged at 2500 rpm for5 minutes at 10° C. to recover the double stranded cDNA (volume ˜50 μl).The cDNA was precipitated with ethanol, resuspended in sterile H₂ O andquantitated by absorption at 260 and 280 nm.

Two hundred pg of double stranded cDNA was used in a 400 μl PCRreaction. The primer set used to prime the PCR was: muDHFR.A: 5'-CGGCAACGCGTGCCATCATGGTTCGAC-3' (SEQ ID NO:16) and muDHFR.B: 5'-CGGCAGCGGCCGCATAGATCTAAAGCCAGC-3' (SEQ ID NO:17). The PCR reaction conditionswere as reported in Saiki et al., Science 239:487 (1988). Briefly, thereaction was run at 94° C. for 1 minute, 55° C. for 1 minute, 72° C. for1.5 minutes and 30 cycles were performed. Taq DNA polymerase wasobtained from Perkin-Elmer and the reaction buffer used was thatrecommended by the manufacturer. The primer pair generates a 671 bpfragment having a MluI site at the 5' end and a NotI site at the 3' end(SEQ ID NO:18; the amino acid sequence encoded by SEQ ID NO:18 is listedin SEQ ID NO:19). The PCR reaction products were digested with MluI andNotI and electrophoresed on a low melting temperature agarose gel(SeaPlaque, FMC). The 671 bp fragment was cut out of the gel and theagarose was removed by digestion with β-Agarase I (NEB) followed byisopropanol precipitation according to the manufacturer's directions.

The 671 bp fragment was inserted into pSSD7 which was digested with MluIand NotI to generate pSSD7-DHFR. The map of pSSD7-DHFR is shown in FIG.12.

EXAMPLE 4 Construction of the Expression Vector pJFE 14ΔIL10

pJFE 14ΔIL10 contains a full length cDNA clone encoding the mouseinterleukin 10 (IL-10) protein under the transcriptional control of theSRα enhancer/promoter. As discussed above, the SRα enhancer/promoter isactive in a broad range of cell types. pJFE 14ΔIL10 is used to directthe expression of the IL-10 gene in transfected cells (i.e., pJFE14ΔIL10 expresses IL-10 as the gene of interest).

a. Construction of pJFE 14ΔIL10

The plasmid pJFE14 [Elliott et al. (1990) Proc. Natl. Acad. Sci USA87:6363]was constructed by combining DNA fragments from the plasmidspSSD, pcDL-SRα296 [Takebe et al. (1988) Mol. Cell. Biol. 8:466] andpCDM8 [Seed (1987) Nature 329:840]. pSSD was cut with HindIII and XhoIand a 2.77 kb fragment was isolated from an agarose gel. pcD-SRα296 wascut with HindIII and Xhol and an ˜640 bp fragment was isolated from anagarose gel. The two gel-purified DNA fragments were ligated together togenerate the plasmid pSRαXSD. pSRαSD was cut with XbaI and NotI and a3.4 kb fragment was isolated from an agarose gel. pCMD8 was cut withXbaI and NotI and a 440 bp fragment was isolated. The 3.4 kb and 440 bpXbaI/NotI fragments were ligated together to generate pJEL14. Aschematic of pJFE14 is shown in FIG. 13.

The ΔIL10 cDNA was generated from a full-length mouse cDNA clone, F115[Moore et al. (1990) Science 248:1230] using the PCR. The pcDSRα-F115clone was linearized with BamHI, which cuts out the cDNA insert. A PCRreaction was run using AmpliTaq™ DNA Polymerase (Perkin Elmer) andbuffer supplied by the manufacturer according to their suggestedconditions. The primers used in the PCR were IL10Δ-5'[5'-ATATATCTAGACCACCATGCCTGGCTCAGCACTG-3' (SEQ ID NO:20)] and IL10Δ-3'[5'-ATTATTGCGGCCGCTTAGCTTTTCATTTTGAT CAT-3' (SEQ ID NO:21)]. The PCRreaction was run at 94° C., 1 min, 72° C., 1 min, 46° C., 1 min for 30cycles. The PCR generated DNA has deleted essentially all of thenon-coding sequences and placed an optimal Kozak sequence just 5' to theinitiator ATG of the IL-10 gene sequences. The PCR generated DNA wasextracted with phenol:CHCl₃ (1:1) and then with CHCl₃. The DNA wasethanol precipitated, pelleted in a microcentrifuge and resuspended inTE. The DNA was cut with XbaI and NotI. pJFE14 was cut with XbaI andNotI. Both digestion mixtures were run on a low melt agarose gel. The550 bp ΔIL10 band and the 3.4 kb pJFE14 band were cut out of the gel andcombined in a tube. The DNAs were co-extracted from the agarose, ligatedtogether and transformed into the bacteria DH5α. Colonies were pickedand the clone pJFE14-ΔIL10 was identified. A schematic map ofpJFE14-ΔIL10 is shown in FIG. 14.

EXAMPLE 5 Construction of pSRαSD5-DRα-DAF

pSRαSD5-DRα-DAF contains a cDNA clone encoding a chimeric mouse DRαgene. In this chimeric protein, the extracellular domain of the DRαprotein is joined to sequences derived from the decay acceleratingfactor (DAF) gene. The DAF sequences provide a glycophosphatidylinositollinkage which allows the chimeric protein to be cleaved from the surfaceof the cell (cell surface expression requires the expression of the DRβchain in the same cell) by treatment of the cell with phospholipase C.

a. Construction of the Phagemid Vector pDAF20

To generate pSRαSD5-DRα-DAF and pSRαSD5-DRβ1-DAF (Example 6), a vectorcontaining sequences encoding a portion of decay accelerating factor(DAF) which anchors DAF to the cell surface via aglycophosphatidylinositol linkage was constructed. pDAF20 wasconstructed as follows.

Two micrograms of pBluescript KS(-) (Stratagene) was cut with EcoRV(NEB). TE buffer was added to such that the final volume was 200 μl.Spermine was added to a final concentration of 1.4 mM and the DNA wasallowed to precipitate for 20 minutes on ice. The precipitated DNA wasthen pelleted by centrifugation for 10 min. in a microcentrifuge and thespermine was washed from the pellet exactly as described [Hoopes andMcClure (1988) Nucleic Acids Res. 9:5493]. Briefly, the pellet wasdispersed in extraction buffer [75% EtOH, 1× Buffer 2 (0.3M sodiumacetate, 0.01M magnesium acetate)] by vortexing; the dispersed pelletwas then left on ice for 1 hour. The pellet was collected bycentrifugation for 10 min. in a microcentrifuge. The pellet was dried atroom temperature and resuspended in 14 μl H₂ O. On ice, 250 ng each ofDAFa (SEQ ID NO:22) and DAFb (SEQ ID NO:23) unphosphorylatedoligonucleotides were added to the resuspended DNA. TheDNA-oligonucleotide mixture was then brought to a final concentration of50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 10 mM DTT and 1 mM rATP in a finalreaction volume of 20 μl. Eighty units of T4 DNA ligase (NEB) was addedand the ligation mixture was placed at 14° C. overnight. The ligationmixture was then heated to 65° C. for 10 min. NaCl was added to a finalconcentration of 50 mM and the DNA was digested with EcoRV (NEB). Analiquot of the DNA was then used to transform competent HB101.

Clones were picked and miniprep DNA was examined by restriction enzymedigestion. A clone, called DAF20, was isolated that has the DAF sequencecloned in the EcoRV site of pBluescript KS(-) with the XbaI at one endof the DAF sequence adjacent to the EcoRI site in the polylinker andaway from the HindIII site in the polylinker. The sequence of the pDAF20polylinker region containing the DAF insert is listed in SEQ ID NO:24.

The resulting plasmid pDAF20 contains DNA encoding the final 37 aminoacids of the form of DAF that is anchored to the cell surface by aglycophosphatidylinositol (PI) linkage [Caras et aL (1987) Nature325:545]. Chimeric proteins containing these 37 amino acids at theirC-terminus, can be expressed on the cell surface of mammalian (andinsect) cells with this PI anchor. This anchor can be readily cleavedand the protein solubilized from the cell surface usingphosphatidylinositol-specific phospholipase C [Caras et al. (1987)Science 238:1280].

Phosphatidylinositol-specific phospholipase C was purified from Bacillusthuringiensis (ATCC 10792) exactly as described [Kupke et al. (1989)Eur. J. Biochem. 185:151]; phosphatidylinositol-specific phospholipase Cis available commercially (e.g., Sigma).

The use of soluble class II molecules complexed with specific peptideshas been suggested for the treatment of autoimmune disease [Sharma, etal. (1991) Proc. Natl. Acad. Sci. USA 88:11465]. Such therapy requiresthat ample quantities of soluble class II molecules be available. Thepresent invention allows large quantities of soluble class II moleculesto be produced from cells expressing class II molecules on the cellsurface wherein these molecules are anchored to the cell via the PIanchor provided by sequences derived from DAF. Alternatively, solubleforms of cell surface proteins can be produced according to the methodsof the present invention using DNA sequences encoding chimeric class IImolecules containing a thrombin cleavage site between the extracellulardomain and the transmembrane domain of each chain comprising the classII heterodimer.

b. Isolation of a Full-Length HLA DRα cDNA

A cDNA library was prepared from poly A⁺ mRNA isolated from IBw4 cells(GM03104B, NIGMS Human Genetic Mutant Cell Repository at the CoriellInstitute for Medical Research, Camden, N.J.) using standard techniques[Sambrook et al., supra at 7.26-7.29]. cDNA was generated from the mRNAand inserted into the cloning vector λgt10 using standard techniques[Huynh et al., in DNA Cloning: A Practical Approach (D. M. Glover, ed.),vol. 1, IRL Press Oxford (1985), pp. 49-78]. A full-length DRα cDNA wasisolated from the library using a partial DRα cDNA as a probe; thepartial DRα cDNA was contained within pDRα1 [Stetler et al. (1982) Proc.Natl. Acad. Sci. USA 79:5966]. The resulting full-length DRα cDNA wascontained on a 1.2 kb NotI/EcoRI fragment.

C. Construction of SRαSD5-DRα-DAF

An in-frame connection between the extracellular coding sequence of DRαand the DAF sequence was performed using site-directed in vitrodeletional mutagenesis [Kunkel et al. (1987) Methods in Enzymology154:367]. The mutational, bridging oligonucleotide encodes the desiredconnection.

The full length DRα cDNA was subcloned as a NotI-EcoRI fragment intopDAF20 (section a above). The pDAF20-DRα was isolated and transformedinto the bacteria BW313 [Kunkel et al. (1987), supra]. A colony was thengrown overnight in LB containing 100 μg/ml ampicillin. The overnightculture was diluted 1:10 in a final volume of 6 ml and grown at 37° C.After 1 hour, 400 μl of a stock of helper phage R408 [Russel et al.(1986) Gene 45:333] having a titer of approximately 1×10¹¹ pfu/ml wasadded to the culture and the culture was grown at 37° C. forapproximately 8 hours. One point four (1.4) ml aliquots of the culturewere then placed into 4 microcentrifuge tubes and spun in amicrocentrifuge 5 min at 4° C. One point one (1.1) ml of eachsupernatant was transferred to fresh microcentrifuge tubes containing150 μl of 20% PEG(6000), 2.5 M NaCl. The contents of the tubes weremixed and allowed to stand at room temp. for at least 20 min.Precipitated, ssDNA containing phage particles were pelleted in amicrocentrifuge for 5 min at 4° C. Care was taken to remove all thePEG-containing supernatant from the pellets. The four pellets wereresuspended in a total of 200 μl of 300 mM NaOAc, pH 7 and extractedwith an equal volume of phenol:CHCl₃ (1:1) twice, and then once withCHCl₃. Two volumes of ethanol was added to the supernatant and chilledto -20° C. The ssDNA was pelleted in a microcentrifuge 20 min at 4° C.The pellet was dried and resuspended in 10 μl TE buffer.

The bridging oligonucleotide was phosphorylated in a volume of 20 μlcontaining 50 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 10 mM DTT, 1 mM rATPand 65 ng of the RADAF2 oligonucleotide (SEQ ID NO:25) with 8 units ofT4 DNA polynucleotide kinase (Pharmacia) at 37° C. for 1 hour. To annealthe bridging oligonucleotide to the ssDNA template, 1.1 μl of thephosphorylated RADAF2 oligonucleotide (SEQ ID NO:25) and 5 μl of thessDNA prep were mixed in a final volume of 15 μl of 40 mM Tris-HCl (pH7.5), 20 mM MgCl₂, 50 mM NaCl, heated to 70° C. and allowed to cool toroom temp. on the bench top. In the reaction tube, the concentrations ofthe buffers were adjusted to give, in a final volume of 95 μl, 16.8 mMTris-HCl, pH 7.5, 11.6 mM MgCl₂, 7.9 mM NaCl, 10.5 mM DTT and 1.1 mMrATP. Four units of T4 DNA ligase (NEB) and 3.8 units of Sequenase (USBiochemicals) were added to the reaction, which was incubated at roomtemp. for 5 min and 37° C. for 1 hour. The reaction was adjusted to 58mM NaCl and heated at 65° C. for 10 min. The tube was cooled to 37° C.and the DNA cut with EcoRi and XbaI. An aliquot of DNA was transformedinto E. coli strain TG2 and plated on ampicillin-containing plates. Aclone that showed the proper deletion of DNA between the desiredconnection of the DRα and DAF sequences was isolated. This clone wassequenced to confirmed the presence of the desired sequences usingstandard techniques. The coding region for the DRα-DAF protein is listedin SEQ ID NO:26; the amino acid sequence encoded by SEQ ID NO:26 islisted in SEQ ID NO:27.

The plasmid containing the correct DRα-DAF construct was cut withHindIII. The ends generated by HindIII digestion were made blunt withKlenow enzyme and unphosphorylated EcoRI linkers were ligated onto theblunt ends using standard techniques. The DNA was transformed intocompetent E. coli and clones which contained the DRα-DAF sequences as aNotI-EcoRI fragment were isolated. The DRα-DAF DNA was then subclonedinto the pSRαSD5 plasmid as a NotI-EcoRI fragment to generatepSRαSD5-DRα-DAF. The map of pSRαSD5-DRα-DAF is shown in FIG. 15.

EXAMPLE 6 Construction of pSRαSD5-DRβ1-DAF

pSRαSD5-DRβ1-DAF contains a cDNA clone encoding a chimeric mouseDRβ1-DAF gene. In this chimeric protein, the extracellular domain of theDRβ1 protein is joined to sequences derived from the DAF gene. The DAFsequences provide a glycophosphatidylinositol linkage which allows thechimeric protein to be cleaved from the surface of the cell (cellsurface expression requires the expression of the DRα chain in the samecell) by treatment of the cell with phospholipase C.

a. Isolation of a Full-Length DRβ1 cDNA

A cDNA library was prepared from poly A⁺ mRNA isolated from IBw4 cells(GM03104B, NIGMS Human Genetic Mutant Cell Repository at the CoriellInstitute for Medical Research, Camden, N.J.) using standard techniques[Sambrook et al., supra at pp. 7.26-7.29]. cDNA was generated from mRNAand inserted into the cloning vector λgt10 using standard techniques[Huynh et al:, in DNA Cloning: A Practical Approach (D. M. Glover, ed.),vol. 1, IRL Press Oxford (1985), pp. 49-78]. A full-length DRPβ1 cDNAclone was isolated from the library using a full length DRβ cDNA probewhich was contained within the plasmid p2918.4 [Bell et al. (1985) Proc.Natl. Acad. Sci. USA 82:3405]. The resulting full-length DRβ1 clone wascontained on a 1.2 kb NotI/EcoRI fragment.

b. Construction of pSRαSD5-DRβ1-DAF

An in-frame connection between the extracellular coding sequence of DRβand the DAF sequence was performed using site-directed in vitrodeletional mutagenesis [Kunkel et aL (1987), supra] as described inExample 5c.

The full length DRβ1 cDNA (section a above) was subcloned into pDAF20(Ex. 5a) as a NotI-EcoRI fragment to generate pDAF20-DRβ1. pDAF20-DRβ1DNA was isolated and transformed into the E. coli strain BW313. A colonywas then grown overnight in LB containing 100 μg/ml ampicillin. Theovernight culture was diluted and incubated with helper phage asdescribed in Example 5c to generate single-stranded pDAF20-DRPβ1 DNA.The ssDNA was precipitated and resuspended in TE buffer as described inExample 5c.

The bridging oligonucleotide, RQBDAF2 (SEQ ID NO:28), was phosphorylatedas described in Example 5c. To anneal the bridging oligonucleotide tothe ssDNA template, 1.1 μl of phosphorylated RADAF2 and 5 μl of thessDNA prep were mixed, heated and cooled as described in Example 5c. Thereaction mixture was adjusted to give, in a final volume of 95 μl, aconcentration of 16.8 mM Tris-HCl (pH 7.5), 11.6 mM MgCl₂, 7.9 mM NaCl,10.5 mM DTT and 1.1 mM rATP. Four units of T4 DNA ligase (NEB) and 3.8units of Sequenase (US Biochemicals) were added to the reaction, whichwas incubated at room temp. for 5 min and 37° C. for 1 hour. Thereaction was adjusted to 58 mM NaCl and heated at 65° C. for 10 min. Thetube was cooled to 37° C. and the DNA digested with EcoRI and XbaI. Analiquot of the digested DNA was used to transform E. coli strain TG2.The transformed cells were plated on plates containing ampicillin. Aclone that showed the proper deletion of DNA between the desiredconnection of the DRβ1 and DAF sequences was isolated. The presence ofthe desired sequences was confirmed by DNA sequencing using standardtechniques. The coding region for the DRβ1-DAF protein is listed in SEQID NO:29; the amino acid sequence encoded by SEQ ID NO:29 is listed inSEQ ID NO:30.

The plasmid containing the correct DRβ1-DAF construct was cut withHindIII. The DNA was blunted with Klenow enzyme and EcoRI linkers wereadded to the blunted ends using standard techniques. The DNA wastransformed into bacteria that contained the DRβ1-DAF as a NotI-EcoRIfragment were isolated. The DRβ1-DAF DNA was subcloned into pSRαSD5 as aNotI-EcoRI fragment to generate pSRαSD5-DRβ1-DAF. The map ofpSRαSD5-DRβ1-DAF is shown in FIG. 16.

EXAMPLE 7 High-Level Expression of Recombinant IL-10 In Lymphoid Cells

High levels of IL-10 were expressed in BW5147.G.1.4 cells (a T lymphoidcell line) by co-amplification of the following three plasmids: 1) theexpression vector pJFE 14ΔIL10 which encodes mouse IL10; 2) theselection vector pMSD5-HPRT which encodes the HPRT enzyme and 3) theamplification vector pSSD7-DHFR which encodes the mouse DHFR enzyme. Theplasmids were introduced into BW5147.G.1.4 cells by electroporation. Theplasmid DNA was isolated from bacterial cells using CsCl densitygradient centrifugation.

The plasmids were prepared for electroporation as follows. First, theplasmids were linearized in the same reaction tube. 200 μg of pJFE14ΔIL10 was digested with SalI. Ten μg of pMSD5-HPRT was digested withSalI. Twenty μg of pSSD7-DHFR was digested with SalI. SalI was obtainedfrom New England BioLabs and restriction digests were performedaccording to the manufacturer's instructions. The linearized plasmidswere then precipitated with ethanol and resuspended in 0.5 ml of 1×HBS(EP) buffer [20 mM HEPES (pH 7.0); 0.75 mM Na₂ HPO₄ /NaH₂ PO₄ (pH7.0); 137 mM NaCl; 5 mM KCl and 1 gm/l dextrose].

BW5147.G.1.4 cells were grown in RPMI 1640 medium (Gibco/BRL) containing10% FCS (HyClone) and 50 μg/ml gentamycin (Sigma). Prior toelectroporation, the cells were washed twice in ice cold 1× HBS(EP)buffer and resuspended at 2×10⁷ cells/ml in 0.5 ml of 1× HBS(EP). Thecells were then placed in a 1 ml cuvette (Sarstedt) which contained thelinearized DNAs in 0.5 ml of 1× HBS(EP). The cuvette was placed on ice.The electroporation was performed at 225 volts using an ISCO Model 493power supply. The electroporation apparatus was constructed exactly asdescribed [Chu, G. et al., (1987) Nucl. Acids Res. 15:1311]. Theelectroporation device was set on constant voltage (225 V) at the 2×setting (i.e., both capacitors were used). Following electroporation,the cells were allowed to recover by incubation on ice for 5 to 15minutes.

The electroporated cells were then transferred to a T75 flask (Falcon)containing 30 ml of RPMI 1640 medium containing 10% FCS and 50 μg/mlgentamycin. The cells were placed in a humidified atmosphere containing5% CO₂ at 37° C. for 36 hours. The cells were then plated in 24 wellplates (Falcon, Lincoln Park, N.J.) at a density of 1×10⁴ cells/well inselective medium [RPMI 1640 containing 10% FCS, 100 μM hypoxanthine(Sigma) and 2 μg/ml azaserine (Sigma)]. Each well contained 0.5 ml ofselective medium. One week after plating the cells in the 24 wellplates, 0.5 ml of fresh selective medium was added.

HPRt⁺ colonies (i.e., wells containing growing cells or positive wells)were visible after approximately 10 days. At day 13 (with the day ofelectroporation being day zero) 100 μl of culture supernatant wasremoved and assayed for the presence of mouse IL10 using an ELISA assayperformed as described [Mosmann et al. (1990) J. Immunol. 145:2938]. Themonoclonal antibody (mcab) SXC1 (PharMingen, San Diego, Calif.) was usedas the capture antibody and biotinylated mcab SXC2 [the mcab JESS-2A5(PharMingen) may be used in place of SXC2] was used as the detectionantibody. Briefly, 20 μl of mcab SXC1 at a concentration of 2 μg/ml inPBS was allowed to bind to the wells of flexible vinyl 96 well plates(Falcon) by incubating for 30 min to 3 hours at 37° C. Excess proteinbinding sites were then blocked by adding 200 μl/well PBS, 10% FCS.After 30 minutes of blocking at 37° C., the plates were washed with PBS,0.1% Tween 20 (ICN Biochemicals, Aurora, Ohio.). Samples to be testedwere added at 50 μl/well and incubated 1 hour at 37° C. Plates werewashed with PBS, 0.1% Tween 20 and 20 μl/well of PBS, 0.1% Tween 20, 1μg/ml biotinylated mcab SXC2 was added. The plates were incubated 30min. at 37° C. The supernatants were removed and the plates were washedwith PBS, 0.1% Tween 20. A 1/5000 dilution of streptavidin-horseradishperoxidase conjugate (Jackson Immunoresearch Laboratories, West Grove,Pa.) in PBS, 0.1% Tween 20, 0.1% BSA was added at 50 μl/well andincubated 30 min. at 37° C. The plates were then exhaustively washedwith PBS, 0.1% Tween 20 and 100 μl/well of 44 mM NaH₂ PO₄, 28 mM CitricAcid, 0.003% H₂ O₂, 1 mg/ml 2,2'azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (Sigma) was added. Theoptical densities (ODs) of the wells were measured after 1 hour using aVMAX microplate reader (Molecular Devices, Menlo Park, Calif.) with atest wavelength of 405 nm and a reference of 490 nm.

The cells from wells containing 1 to 3 apparent clones and whichcontained greater than or equal to 100 units IL10/ml were cloned bylimiting dilution using standard techniques [Cloning by LimitingDilution, in Current Protocols in Immunology (J. E. Coligan et al.,eds.) John Wiley & Sons, New York, section 2.5.10]. For the limitdilution cloning, the cells were plated at 2 cells or 4 cells per wellin a 96 well plate (Falcon) in selective medium; one 96 well plate wasset up for each cell density (2 or 4 cells/well). In total, 16independent colonies were cloned by limit dilution.

Eight days after limit dilution cloning was initiated, isolated colonieswere picked from each of the limit dilution plates; these colonies weretransferred to a 96 well plate; each well contained 5 ml RPMI 1640containing 10% dialyzed FCS (HyClone) and 100 μM hypoxanthine. The useof dialyzed serum at this point increases the speed and frequency ofamplification of the transfectants; hypoxanthine is added to the mediumat this point as it is required for the growth of the cells for a fewpassages until the azaserine level is diluted to a negligibleconcentration.

Two days later, 100 μl of culture supernatant was tested for thepresence of IL-10 using an ELISA as described above. The twobest-producing clones from each of the original wells (e.g., the 24 wellplate) were chosen for further manipulation. In total 19 clones (termedselectants as these clones have survived growth in selective medium buthave not yet been subjected to amplification by growth in the presenceof methotrexate) were chosen.

Five days after the transfer of the isolated colonies (cloned by limitdilution) to 96 well plates, the colonies were transferred to 24 wellplates and allowed to expand. The expanded colonies were thentransferred to 5 ml flasks (Falcon) containing 5 ml of RPMI 1640 mediumcontaining 10% dialyzed FCS. The clones produced between 100 and 200units/ml of IL-10.

The selected clones were then subjected to amplification by growing thecells in the presence of methotrexate. The 19 clones were each testedfor their sensitivity to methotrexate (MTX). Five×10⁴ cells from eachclone was placed into a well in a series of 24 well plates. The cloneswere grown in the presence of RPMI 1640 medium containing 10% dialyzedFCS and either 3, 10, 30, 60 or 90 nM MTX. Six clones were able to growin the presence of greater than or equal to 30 nM MTX; these clones wereretained.

The six clones resistant to ≧30 nM MTX were plated in T25 flasks(Falcon) containing 5 ml of RPMI 1640 medium containing 10% dialyzed FCSand either 90, 150 or 210 nM MTX. Three flasks were set up for eachclone. The clones were allowed to grow for 15 days at these threeconcentrations of MTX and then supernatants were taken from each flaskand assayed for IL-10 production using an ELISA as above. All clonesfrom flasks containing 90 or 150 nM MTX produced between 800 and 1200units/ml of IL10. The best producing clone from each of the six originalMTX^(r) clones was selected (one from a 90 nM MTX flask and the restfrom 150 rM MTX flasks). These clones were then expanded to 5 mls inmedium containing the appropriate concentration of MTX (over a 6 dayperiod). The clones were then transferred into medium containing either450, 750 or 1050 nM MTX. Sixteen days later supernatants from clonesgrowing in the presence of 1050 nm MTX were assayed for IL-10production. The clones were found to produce between 12,000 and 76,000units/ml of IL-10 (one clone produced 12,000 u/ml, one clone produced15,000 u/ml and eight clones produced between 50,000 and 76,000 u/ml).

The two clones producing the highest levels of IL-10 were chosen; theseclones were designated as 9-2 and 11-2. Clones 9-2 and 11-2 were thengrown in the presence of 5 μM MTX for 3 weeks, expanded and then frozen.Cultures were frozen as follows. Thirty milliliters of media containingcells at a density of 6 to 10×10⁵ cells per ml were pelleted in a 50 mlconical tube (Falcon) at 500×g for 5 minutes. The supernatant was pouredoff and the cells were resuspended in 7.5 ml of Freezing Media (40% FCS,53% RPMI 1640, 7% DMSO) and placed in 5 freezing vials (Nunc,Naperville, Ill.). The cells were placed in a -70° C. freezer for 24 to96 hours and then transferred to liquid nitrogen for long term storage.

Aliquots of each clone were thawed after approximately 2 months,re-tested for IL-10 production and grown continuously in the presence of5 μM MTX. These two clones (9-2 and 11-2) continue to produce between64,000 to 86,000 units/ml of IL10.

The levels of expression of IL10 were roughly equivalent when the cellswere grown at 1 or 5 μM MTX (compare 76,000 at 1 μM to 64-86,000 at 5μM). The use of concentrations of MTX greater than 5 μM appeared to makethe cells grow more slowly so that the total yield of protein was nogreater than that obtained by growing the cells in the presence of 1 to5 μM MTX.

It should be noted that selective pressure to maintain the expression ofthe HPRT protein (i.e., growth in the presence of medium containinghypoxanthine and azaserine) was not used after the cells weretransferred into medium containing MTX with no loss of IL-10 expression.Furthermore, because the level of IL-10 continued to rise withincreasing concentrations of MTX, the endogenous DHFR gene is not likelyto be amplified in the MTX^(r) cells. In other words, the increase inMTX-resistance is due to the amplification of the exogenous DHFR genepresent on the amplification vector pSSD7-DHFR.

EXAMPLE 8 High-Level Expression of DR Class II MHC in Lymphoid Cells

High levels of DR class II MHC molecules were expressed on the surfaceof BW5147.G.1.4 cells by co-amplification of the following fourplasmids: 1) the expression vector pSRαSD5-DRα-DAF which encodes thealpha chain of the human DR molecule linked to a DAF tail; 2) theexpression vector pSRαSD5-DRβ1-DAF which encodes the beta chain of thehuman DR molecule linked to a DAF tail; 3) the selection vectorpMSD5-HPRT which encodes the HPRT enzyme and 3) the amplification vectorpSSD7-DHFR which encodes the mouse DHFR enzyme. The plasmids wereintroduced into BW5147.G.1.4 cells by electroporation. The plasmid DNAswere isolated from bacterial cells using the standard technique of CsCldensity gradient centrifugation.

The isolated plasmid DNAs were prepared for electroporation as follows.First the plasmids were linearized in the same reaction tube. All fourplasmids were linearized with SalI. The following amounts of plasmidwere used: 200 μg of pSRαSD5-DRα-DAF; 200 μg of pSRαSD5-DRβ1-DAF; 10 μgof pMSD5-HPRT and 25 μg of pSSD7-DHFR. The linearized plasmids were thenprecipitated with ethanol and resuspended in 0.5 ml of 1× HBS(EP)buffer.

BW5147.G.1.4 cells were grown in RPMI-1640 medium containing 10% FCS and50 μg/ml gentamicin. Prior to electroporation the cells were washedtwice in ice cold 1× HBS(EP) buffer and resuspended at a density of2×10⁷ cells/ml in 0.5 ml of 1× HBS(EP). The cells were then placed in a1 ml cuvette (Sarstedt) which contained the linearized DNAs in 0.5 ml of1× HBS(EP). The cuvette was placed on ice. The electroporation wasperformed as described above.

After electroporation the cells were allowed to recover by incubation onice and then they were placed in a T75 flask (Falcon) containing 30 mlof RPMI-1640 medium containing 10% FCS and 50 μg/ml gentamicin. Thecells were placed in a humidified atmosphere containing 5% CO₂ at 37° C.and grown in bulk culture for 36 hours. The cells were then plated intofour 48 well plates (Costar) at a density of 10⁴ cells/well in 0.5 mlselective medium [RPMI 1640 containing 10% FCS, 100 μM hypoxanthine(Sigma) and 2 μg/ml azaserine (Sigma)]. The use of a cell density of1×10⁴ ensures that any colonies which arise are derived from a singlecell; that is this density provides for limit dilution cloning. Anyremaining cells were plated at a density of 1×10⁵ cells/well in 0.5 mlof selective medium. One week after plating in the 48 well plates anadditional 0.5 ml of selective medium was added.

Wells containing clones capable of growth in the selective medium(selectants) were visible after 8 days. Positive colonies (i.e.,positive for growth in selective medium) were picked into 12 well plates(Costar) containing 4 ml of RPMI 1640 containing 10% dialyzed FCS(HyClone) and 100 μM hypoxanthine 10-12 days after the application ofselective medium. The use of dialyzed serum at this point increases thespeed and frequency of amplification of the selectants; hypoxanthine isadded to the medium at this point as it is required for the growth ofthe cells for a few passages until the azaserine level is diluted to anegligible concentration. The cells were allowed to grow for 3-4 days inthe 12 well plates.

Colonies which grew in the presence of hypoxanthine and azaserine(selectants) were checked for the ability to express the DR molecule onthe surface of the cell by staining cells with the monoclonal antibodyL243. L243 binds specifically to the human HLA-DR antigens [Lampson andLevy, J. Immunol., 125:293 (1980)].

The antibody was prepared as follows. Hybridoma L243 was grown and theculture supernatant collected using standard techniques [Harlow andLane, eds., Antibodies: A Laboratory Manual, Cold Spring Harbor Press,New York (1988), pp. 272, 276]. The monoclonal antibodies were purifiedfrom the hybridoma supernatants. L243 was purified on a ProteinA-Sepharose column (Pharmacia) using the protocol supplied by themanufacturer. The purified monoclonal antibody was then biotinylatedusing standard techniques [Antibodies: A Laboratory Manual, supra at p.341]. Biotin was obtained from Vector. Biotinylated L243 was used at adilution of 1:200.

The cells were stained as follows. The contents of the wells on the 12well plates were gently mixed by pipeting the medium. One to 2 ml of thecell suspension was removed; this sample size contains 1-3×10⁶ cells.The cells were pelleted by centrifugation at 1000 rpm for 4 minutes at4° C. One hundred μl of L243 diluted into staining media (10 mM HEPES,pH 7.0, 5% calf serum, 4 mM sodium azide in Hanks balanced saltsolution) was added. The cells were incubated for 20 minutes on ice. Thecells were then washed by adding 1 ml of staining media and then thecells were underlaid with 1 ml of calf serum. The cells were pelletedthrough the serum by centrifugation at 1000 rpm for 4 minutes at 4° C.The supernatant was removed by aspiration. The cells were then suspendedin 100 μl of fluorescein isothiocyanate (FITC) conjugated avidin(Vector, used at 1:50 dilution). The cells were incubated for 20 minuteson ice. The cells were then washed as described above.

The supernatant was removed and the cells were suspended in 200 μl ofstaining media containing 2 μg/ml propidium iodide. Propidium iodide isexcluded from living cells but taken up by dead or dying cells. Theaddition of propidium iodide allows the exclusion of dead cells(propidium iodide-bright cells) from the analysis. The cells werefiltered through nylon screen (Nitex nylon monofilament, 48 micron mesh,Fairmont Fabrics, Hercules, Calif.) prior to analysis on a FACScan™(Becton-Dickinson). An aliquot of parental BW5147.G.1.4 cells (i.e., nottransfected) was stained as above to provide a negative control.

FIG. 17 shows the results of staining a representative selectant clone,clone 5, with L243. FIG. 17 is a histogram showing the log offluorescein (x axis) plotted against the relative number of cells in thesample. Cells which express the DR molecule on the surface of theBW5147.G.1.4 cell appear as fluorescein bright cells due to staining ofthe cell surface with biotylinated-L243 followed by FITC-avidin. Asshown in FIG. 17, all of the cells in clone 5 express the transfected DRmolecule. The fact that surface expression of the DR molecule is seenshows that both the α and the β chain DR constructs are expressed insideclone 5.

Eight selectant clones having the highest levels of expression of DRwere chosen for further manipulation. These eight selectant clones werethen tested for their sensitivity to MTX. Each clone was plated at adensity of 2×10⁴ cells/well in a 24 well plate. Each well contained 1 mlof medium containing RPMI-1640, 10% dialyzed FCS and MTX. The cloneswere grown in the presence of either 3, 10, 30, 60 or 90 nM MTX.Non-transfected BW5147.G.1.4 cells were also grown in the above range ofMTX as a control. Clones which grew in MTX levels at least 2-3 foldhigher than that tolerated by the parental BW5147.G.1.4 (typically lessthan or equal to 10 nM MTX) were selected for further analysis. Four ofthe selectant clones grew in greater than or equal to 30 nM MTX and wereretained; these clones are the primary transfectants chosen foramplification. All 4 clones which grew in >30 nM MTX were analyzed forthe ability to express DR molecules on the surface by an ELISA. The cellsurface ELISA was performed as follows.

Between 5 and 20×10⁴ cells/well were put into a U-bottom 96 well plate.The cells were pelleted in a centrifuge using a plate carrier at 1000rpm for 3 min at 4° C. The supernatant was flicked from the wells, thecells dispersed from their pellets by tapping and the plate was placedon ice. Fifty microliters of a 1/200 dilution of biotinylated mcab L243(Becton-Dickinson) in staining media [Hank's Basic Salt Solution (IrvineScientific), 10 mM HEPES, pH 7, 5% calf serum] was added to each well.The cells were incubated with the biotinylated mcab for 20 min on ice.Ice cold staining media was added to a final volume of 200 μl/well. Thecells were pelleted and the supernatant flicked out and the pelletsdispersed as described above. The cells were washed twice more with 200μl/well of ice cold staining media. Fifty microliters of a 1/1000dilution of Horseradish peroxidase conjugated Avidin (VectorLaboratories, Burlingame, Calif.) was added per well and incubated onice for 20 min. Ice cold staining media was added to a final volume of200 μl/well. The cells were pelleted and the supernatant flicked out andthe pellets dispersed as described above. The cells were washed threemore with 200 μl/well of ice cold staining media. After the final wash,the plate was again tapped to disperse the cell pellets and each wellreceived 200 μl of freshly made OPD Substrate Solution [16 mM CitricAcid, 34 mM Sodium Citrate, 0.01% H₂ O₂, 1 mg/ml O-phenylene diaminedihydrochloride (Sigma)]. The plate was allowed to sit at room temp for10 to 20 min. The cells were then pelleted at 1000 rpm for 3 min at 4°C. One hundred microliters of supernatant from each well was transferredto a fresh, flat bottom 96 well plate (Costar) and the plate was read ona VMAX microplate reader (Molecular Devices, Menlo Park, Calif.) at awavelength of 450 nm.

All four clones expressed the DR molecule as judged by ELISA analysis.Each of these four clones was grown in the highest MTX level at whichobvious growth still occurred as determined by the test for MTXsensitivity above; the levels ranged from 30 to 80 nM MTX. The cloneswere then again checked for the ability to express DR on the cellsurface by staining with L243 and FACS analysis as above. One out fourfirst round amplificants, clone 5, showed both an increased resistanceto MTX and the best corresponding increase in DR expression (all fourclones showed increased DR expression). The histogram of cells fromclone 5 grown in 80 nM MIX is shown in FIG. 18. In FIG. 18 the log offluorescein (x axis) is plotted against the relative number of cells inthe sample. Growth in 80 nM MTX represents the first round ofamplification for clone 5.

The three clones which grew in higher levels of MTX but which did notshow a high coincidental increase in the expression of DR werediscarded. Clone 5 was retained and subjected to further rounds ofamplification by grow in increasing concentrations of MTX. FIGS. 19 and20 show histograms of cells from clone 5 grown in 320 nM and 1 μM MTX,respectively. The cells were stained with L243 and analyzed on a FACScanas described above. As is shown in FIGS. 19 and 20, clone 5 continued toshow a coincidental increase in DR expression and increasedMTX-resistance. Integration of the area under the peaks of fluorescencefrom each of FIGS. 17-20 showed that clone 5 achieved a 30-fold increasein DR expression between the initial selectant stage and the third roundof amplification (1 μM MTX^(r)).

Continued analysis of clone 5 demonstrated that it is extremely stable.Clone 5 grown in 1 μM MTX (referred to as the 1 μM MTX amplificant ofclone 5) can be grown for 2 to 3 weeks in medium lacking MTX without anyapparent drop in expression of DR (as judged by cell surface ELISAassays).

EXAMPLE 9 Production of Large Quantities of Soluble T Cell Receptor andClass II MHC Molecules

Tumors of B and T cells (i.e., lymphomas and leukemias) are often clonalin nature and therefore the Ig or TCR carried on the surface of thetumor cell can serve as a tumor-specific antigen. Soluble forms of thetumor-specific Ig have been used to immunize patients in order to invokean immune response against the tumor cell [Kwak et al. (1992) N. Engl.J. Med. 327:1209 and Hsu et al. (1996) Nature Med. 2:52]. Thetherapeutic use of soluble forms of a patient's tumor-specific antigenrequires that large quantities of the soluble antigen be produced in ashort period of time so that immunization of the patient can be carriedout quickly (i.e., before the patient's disease progress to a point thattherapy is pointless). Large quantities of soluble class II MHCmolecules are required to allow treatment of autoimmune disease usingsoluble class II molecules complexed with specific peptides [Sharma, etal. supra].

The methods of the present invention allow the production of largequantities of soluble forms of class II MHC molecules and TCR to beproduced in a rapid manner. These methods allow for the production ofcustomized tumor cell vaccines comprising soluble TCR for the treatmentof lymphoma and leukemia patients as well as the production of solubleclass II MHC molecules for the treatment of autoimmune disease. DNAsequences encoding the chains comprising the extracellular domains ofthe TCR or class II MHC molecules expressed by the patient's tumor cellsare cloned using the PCR. These sequences are joined to sequencesencoding a thrombin cleavage site followed by the transmembrane andcytoplasmic domains of either the α or β chain of a mammalian class IIMHC heterodimer. The sequences encoding each chain of the chimeric TCRor class II MHC molecules (i.e., the genes of interest) are insertedinto any of the SD7 vectors described herein (e.g., pSRαSD7; Ex. 1) andthe resulting vectors are co-transfected into BW5147.G.1.4 cells alongwith an amplification vector (e.g., pSSD7-DHFR; Ex. 3) and, if sodesired, a selection vector (e.g., pMSD5-HPRT; Ex. 2). The transfectedcells will express the chimeric TCR or class II MHC molecules on thecell surface. The transfected cells are subjected to selection and/oramplification in order to produce amplified cell lines which expresslarge quantities of the chimeric TCR or class II MHC molecules on thecell surface. These chimeric proteins can be cleaved from the cellsurface to produce soluble TCR or class II MHC molecules by digestionwith thrombin.

The following discussion illustrates the production of soluble TCR orclass II MHC proteins using amplified cell lines. An analogous approachcan be used to produce soluble forms of any multi-chain cell surfaceprotein.

a. Construction of Vectors Encoding Chimeric TCR Chains

Sequences encoding chimeric α chain of a TCR are constructed whichcomprise (from the amino- to carboxyl-termini) the extracellular domainsof the a chain of a TCR followed by 21 amino acids derived from thethrombin receptor which comprise a thrombin cleavage site followed by 41amino acids comprising the transmembrane and cytoplasmic domains of theclass II MHC molecule DRα. An analogous construct is used to construct achimeric β chain of a TCR comprising (from the amino- tocarboxyl-termini) the extracellular domains of the β chain of a TCRfollowed by 21 amino acids derived from the thrombin receptor whichcomprise a thrombin cleavage site followed by 42 amino acids comprisingthe transmembrane and cytoplasmic domains of the class II MHC moleculeDRβ1. Any mammalian class II MHC αβ pair can be used to providesequences encoding the transmembrane and cytoplasmic domains of the MHCmolecule which permit the association of the chimeric TCR chains. While,the number of amino acid residues comprising the transmembrane andcytoplasmic domains of the α and β chains of the class II MHC moleculesdiffers by one, both MHC junctions are at the third amino acid residuefrom the beginning of the transmembrane domain. This arrangementpreserves the glutamate residue from the α chain and the lysine from theβ chain which have been shown to have a positive effect upon heterodimerformation of class II MHC molecules [Cosson and Bonifacino (1992)Science 258:659].

A vector containing sequences encoding the thrombin and class II MHCsequences is constructed by synthesizing the DNA sequences listed in SEQID NO:31 and SEQ ID NO:33. The amino acid sequence encoded by SEQ IDNO:31 is listed in SEQ ID NO:32 and amino acid sequence encoded by SEQID NO:33 is listed in SEQ ID NO:34.

SEQ ID NO:31 encodes the thrombin site-DRα chimeric sequence and SEQ IDNO:33 encodes the thrombin site-DRβ1 chimeric sequence. Inspection ofthese sequences shows that the sequences at the 5' end which encodes thethrombin site contains the recognition site for the followingrestriction enzymes: BamHI, PvuI and FspI. A NotI site is located at the3' end of the thrombin site-DRβ₁ chimeric sequences. The synthetic DNAis inserted into any suitable vector (e.g., pUC 18 or pUC 19) as aBamHI-NotI fragment. The thrombin site encoded by these sequences isvery efficiently cleaved by thrombin due to the presence of thehirudin-like domain following the thrombin cleavage site [Vu et al.(1991) Cell 64:1057 and Vu et al. (1991) Nature 353:674].

DNA sequences encoding TCR chains are isolated from double-stranded EDNAgenerated from a cell line or a patient's tumor (double-stranded cDNAmay be generated using the protocol set forth in Example 3; oligo d(T)may be used to prime first strand cDNA synthesis in place of the SBNSSdTprimer). The double stranded cDNA is then used in PCRs which containprimer pairs designed to amplify either the α chain or the β chain ofthe human TCR. The PCR is conducted using 1 unit/100 μl reaction Pfupolymerase (Stratagene) in the reaction buffer provided by Stratagene, 5ng/100 μl of a cloned template or 25 ng/100 μl of ds-cDNA derived frompolyA+ RNA isolated from a cell line or tumor, 0.1 mM of each of thefour dNTPs and 0.5 μM of each primer. The PCR is cycled at 94° C. for 15sec followed by 60° C. for 30 sec followed by 75° C. for 2 min for 21cycles.

The 5' primer used to amplify TCR sequences contains the followingrestriction sites at the 5' end of the primer: XbaI, EcoRI and MluIfollowed 18-21 nucleotides comprising a consensus sequence derived fromthe V regions of human TCRs.

Therefore the 5' primer will comprise sets of degenerate primers havingthe following sequence: 5'-TCTAGAATTCACGCGT(N)₁₈₋₂₁ -3' (SEQ ID NO:80,where N is any nucleotide and the 18-21 nucleotide stretch represents aconsensus V region sequence. The following 3' primer is used inconjunction with the above-described consensus 5' primer to amplify theextracellular domains of human TCR α chains:5'-CGATCGTGGATCCAAGTTTAGGTTCGTATCTGTTTCAAA-3' (SEQ ID NO:35). The 3'connection for the TCR a chain is made after the asparagine whichappears at position 110 of the constant (C) region of the a chain. Thefollowing 3' primer is used in conjunction with the above-describedconsensus 5' primer to amplify the extracellular domains of human TCR βchains: 5'-CGATCGAGGATCC AAGATGGTGGCAGACAGGACC-3' (SEQ ID NO:36). The 3'connection for the TCR α chain is made after the isoleucine whichappears at position 147 of the C region of the β chain. These 3' primersare designed such that in both cases (i.e., for both the a and the βchain of the TCR) the connection between the extracellular domains ofthe TCR with the thrombin site is made at the fourth amino acid residuefrom the apparent beginning of the respective transmembrane regions ofthe TCR chains. Both 3' primers contain recognition sites for PvuI andBamHI at their 5' ends. The restriction sites located at the 5' ends ofthe primers allows the resulting PCR products comprising a TCR chain tobe removed as a XbaI or EcoRI or MluI (5' end)-BamHI or PvuI (3' end)fragment and joined with the appropriate thrombin-transmembrane DNAsequence [as a BamHI or PvuI (5' end)-NotI (3' end) fragment] andinserted into any of the SD7 vectors (e.g., pSRαSD7). The resultingexpression vectors (one for each of the α chains and the β chains of thechimeric TCR) are co-transfected using electroporation into BW5147.G.1.4cells along with the amplification vector pSSD7-DHFR (Ex. 3) and theselection vector pMSD5-HPRT (Ex. 2). The amount of each plasmid DNA tobe used (the plasmids are linearized before electroporation), theconditions for electroporation, selection and amplification aredescribed above. The resulting amplified cell lines will express thechimeric TCR heterodimer on the surface of the cell. The TCR issolubilized by digestion of the cells with thrombin. The thrombinsolubilized extracellular domains will have 3 (TCR β) or 4 (TCR α) novelamino acids at the C-termini.

b. Construction of Vectors Encoding Chimeric Class II MHC Chains

Sequences encoding a chimeric α chain of a class II MHC protein areconstructed which comprise (from the amino- to carboxyl-termini) theextracellular domains of the α chain of DRα followed by 21 amino acidsderived from the thrombin receptor which comprise a thrombin cleavagesite followed by 41 amino acids comprising the transmembrane andcytoplasmic domains of the class II MHC molecule DRα. An analogousconstruct is used to construct a chimeric β chain of a class II MHCprotein comprising (from the amino- to carboxyl-termini) theextracellular domains of the β chain of DRβ₁ followed by 21 amino acidsderived from the thrombin receptor which comprise a thrombin cleavagesite followed by 42 amino acids comprising the transmembrane andcytoplasmic domains of the class II MHC molecule DRβ₁.

Sequences encoding the extracellular domains of the α and β chains of aclass II MHC heterodimer are isolated using the PCR as described abovewith the exception that the following primer pairs are used in the PCR.Sequences encoding the extracellular domain of DRα are amplified using5'-ACGCGTCCACCATGGCC ATAAGTGGAGTCCCT-3' (SEQ ID NO:37) (this primercontains a MluI site at the 5' end) and5'-GGATCCAACTCTGTAGTCTCTGGGAGAG-3' (SEQ ID NO:38) (this primer containsa BamHI site at the 5' end). The use of these primers allows theconnection of the extracellular domain of DRα with the thrombinsite-transmembrane sequences (described above) after amino acid 191, aglutamate residue in the mature (i.e., after the removal of the signalsequence) DRα protein.

Sequences encoding the extracellular domain of DRβ₁ are amplified using:5'-ACGCGTCCACCATGGTGTGTCTGAAGCTCCTG-3' (SEQ ID NO:39) (this primercontains a MluI site at the 5' end) and 5'-GGATCCAACTTGCTCTGTGCAGATTCAGA-3' (SEQ ID NO:40) (this primer contains a BamHI site at the 5'end). The use of these primers allows the connection of theextracellular domain of DRβ with the thrombin site-transmembranesequences (described above) after amino acid 198, a lysine residue, inthe mature DRβ protein.

The restriction sites located at the 5' ends of the primers allows theresulting PCR products comprising the class II MHC chains to be removedas a MluI (5' end)-BamHI (3' end) fragment and joined with theappropriate thrombin-transmembrane DNA sequence [as a BamHI (5'end)-NotI (3' end) fragment] and inserted into any of the SD7 vectors(e.g., pSRαSD7). The resulting expression vectors (one for each of the αchains and the β chains of the chimeric class II MHC protein) areco-transfected using electroporation into BW5147.G.1.4 cells along withthe amplification vector pSSD7-DHFR (Ex. 3) and the selection vectorpMSD5-HPRT (Ex. 2). The amount of each plasmid DNA to be used (theplasmids are linearized before electroporation), the conditions forelectroporation, selection and amplification are described above. Theresulting amplified cell lines will express the chimeric class IIheterodimer on the surface of the cell. The class II MHC heterodimer issolubilized by digestion of the cells with thrombin.

EXAMPLE 10 Production of Custom Multivalent Vaccines for the Treatmentof Lymphoma and Leukemia

The existing approach toward vaccination (ie., active immunotherapy) ofB-cell lymphoma and leukemia involves the production of a custom vaccinecomprising autologous immunoglobulin idiotype which corresponds to themost abundant antibody molecule expressed on the surface of the B-celltumor. An analogous approach for the treatment of T-cell lymphomas andleukemias would involve the production of a custom vaccine comprisingautologous T cell receptor (TCR) idiotype which corresponds to the mostabundant TCR molecule expressed on the surface of the B-cell tumor.

Existing methods for the production of custom vaccines for the treatmentof B-cell lymphoma employ the "rescue fusion" technique. The rescuefusion technique involves the removal of lymphoma cells by surgicalbiopsy. The tumor cells are then fused with the heterohybridoma cellline K6H6/B5 which has lost the ability to secrete endogenous Ig. Hybridcells which secrete Ig corresponding to the immunophenotype of the tumorsample are expanded and the secreted Ig is purified for use as a vaccine[Kwak et al. (1992), supra]. The Ig produced by rescue fusion representsa single Ig derived from the patient's tumor; this Ig is presumably thepredominant Ig expressed by the tumor. Thus, vaccines produced by rescuefusion are monovalent and do not represent the full complexity of Igexpressed by tumors which contain somatic variants.

In order to produce multivalent custom vaccines from small numbers ofcells quickly and efficiently, the gene amplification techniquesdescribed in the preceding examples are employed. In this example,methods for the production of tumor-specific Ig derived from a B-celllymphoma patient are provided. However, the general approach outlinedherein is applicable for the production of tumor-specific proteinsgenerally (i.e., production of soluble TCR for treatment of T celltumors, production of Ig for treatment of B cell leukemias, etc.).

In this novel approach, the variable regions corresponding to thepatient's Ig (V_(H) and V_(L)) are molecularly cloned and joined to anappropriate constant region gene contained within an expression vector.Expression plasmids containing the patient's V_(H) region(s) joined toeither a Cγ3 or Cγ4 sequence and expression plasmids containing thepatient's V_(L) region(s) joined to either a Cκ or Cλ2 sequence arecotransfected (via electroporation) along with the selectable andamplifiable marker pM-HPRT-SSD9-DHFR into the desired cell line (e.g.,BW5147.G.1.4). The transfected cells are then subjected to selection andamplification as described in the preceding examples. The methodoutlined below permits the production of a multivalent vaccine whichreflects the degree of somatic variation found within the patient'stumor. These novel multivalent vaccine preparations provide superiorvaccines for the treatment of B-cell lymphoma and should reduce the rateof relapse observed when the current generation of monovalent vaccinesare employed.

a) Construction of Expression and Selection/Amplification Plasmids

For the following constructions, unless otherwise stated, all enzymesare obtained from New England Biolabs (NEB) and used in conjunction withthe buffers and reaction conditions recommended by the manufacturer.

i) Construction of pSRαSD9

Two micrograms of pSRαSD7 (Ex. 1) is cut with SalI and HindIII (NEBenzymes, buffers & conditions). The plasmid is spermine precipitated(Ex. 5) and resuspended in 34 μl H₂ O and 4 μl of 10× T4 DNA ligasebuffer. Equal molar amounts (6.3 ng each) of the unphosphorylatedoligonucleotides SXAPH5 (SEQ ID NO:42) and SXAPH3 (SEQ ID NO:43) areadded. The reaction is chilled on ice, 400 units of T4 DNA ligase isadded and the tube is placed at 14° C. overnight. The ligation istransformed into bacteria and clones screened for the presence of theadded AscI & PacI restriction sites. The resulting plasmid is calledpSRαSD9. FIG. 21 provides a schematic map of pSRαSD9.

ii) Construction of pSRαSD9CG3C, pSRαSD9CG4C, pSRαSD9CKC and pSRαSD9CL2C

The plasmids pSRαSD9CG3C, pSRαSD9CG4C, pSRαSD9CKC and pSRαSD9CL2Ccontain sequences encoding the Cγ3, Cγ4, Cκ or Cλ2 constant regions,respectively. The constant regions contained within these expressionvectors are encoded by synthetic DNA sequences which encode the sameamino acid sequences as that found in the native proteins; however, theDNA sequences have been modified to utilize codons which are found mostfrequently in highly expressed mammalian proteins [Haas et al. (1996)Curr. Biol. 6:315 and Zolotukhin et al. (1996) J. Virol. 70:4646]. TheDNA sequence encoding the Cγ3 region is listed in SEQ ID NO:44; theamino acid sequence encoded by SEQ ID NO:44 is listed in SEQ ID NO:45.The DNA sequence encoding the Cγ4 region is listed in SEQ ID NO:46; theamino acid sequence encoded by SEQ ID NO:46 is listed in SEQ ID NO:47.The DNA sequence encoding the Cκ region is listed in SEQ ID NO:48; theamino acid sequence encoded by SEQ ID NO:48 is listed in SEQ ID NO:49.The DNA sequence encoding the Cλ2 region is listed in SEQ ID NO:50; theamino acid sequence encoded by SEQ ID NO:50 is listed in SEQ ID NO:51.

Double stranded DNA corresponding to SEQ ID NOS:44, 46, 48 and 50 aresynthesized (Operon Technologies). Each synthetic DNA sequence is cutwith NotI and BglII, run through a 0.8% SeaPlacque Agarose gel (FMC) andrecovered using β-agarase as described below. Each C region sequence isligated to the two DNA restriction fragments generated from pSRαSD9 asfollows. A 2 μg aliquot of pSRαSD9 is cut with HindIII and BamHI and a2314 bp band is isolated. A second 2 μg aliquot of pSRαSD9 is cut withHindIII and NotI and an 854 bp band is isolated. These fragments areisolated by running each digest on a 0.8% SeaPlacque Agarose (FMC), theappropriate bands are cut out and combined in a microfuge tube. Theagarose is remove by β-Agarase (NEB) digestion and the DNA is recoveredby isopropanol precipitation exactly as indicated by NEB.

The ligation of SEQ ID NO:44 (digested with NotI and BglII) with theabove fragments of pSRαSD9 generates pSRαSD9CG3C (map shown in FIG. 22).The ligation of SEQ ID NO:45 (digested with NotI and BglII) with theabove fragments of pSRαSD9 generates pSRαSD9CG4C (map shown in FIG. 23).The ligation of SEQ ID NO:46 (digested with NotI and BglII) with theabove fragments of pSRαSD9 generates pSRαSD9CKC (map shown in FIG. 24).The ligation of SEQ ID NO:47 (digested with NotI and BglII) with theabove fragments of pSRαSD9 generates pSRαSD9CL2C (map shown in FIG. 25).

iii) Construction of pM-HPRT-SSD9-DBFR

pM-HPRT-SSD9-DHFR contains the hprt gene under the control of theMoloney enhancer/promoter and the dhfr gene under the control of theSV40 enhancer/promoter. pM-HPRT-SSD9-DHFR is constructed by firstsubcloning the HPRT cDNA (Ex. 2) into pMSD8 (described below) to createpMSD8-HPRT. The small DNA fragment located between the SalI and HindIIIsites on pMSD8-HPRT is then replaced with a sequence containing AscI andPacI sites as follows. pMSD8-HPRT is digested with SalI and HindIII andthe SXAPH5 and SAXPH3 oligonucleotides (SEQ ID NOS:42 and 43) areligated to the ends of the digested pMSD8-HPRT (as described in sectioni above) to create pMSD9-HPRT. The ˜2450 bp SalI-ClaI fragmentcontaining the AscI and PacI sites, the Moloney enhancer/promoter, theHPRT cDNA and the EF1α poly A region is inserted between the SalI andClaI sites of pSSD7-DHFR (Ex. 3) to generate pM-HPRT-SSD9-DHFR. FIG. 26provides a map of pM-HPRT-SSD9-DHFR.

pMSD8 is similar to pMSD5 but contains the poly A site from the humanelongation factor 1α gene. pMSD8 was constructed as follows: A 292 bpfragment containing the poly A site from the human elongation factor 1αgene was isolated from MOU cell (GM 08605, NIGMS Human Genetic MutantCell Repository, Camden, N.J.) genomic DNA using PCR. MOU genomic DNAwas isolated using conventional techniques. The PCR was conducted using10 μg MOU genomic DNA and 1 μM fmal concentration of each primer in a400 μl reaction. Reaction conditions were 94° C. for 1 minute, 60° C.for 1 minute, 72° C. for 1.5 minutes, 30 cycles. Taq DNA polymerase wasobtained from Perkin-Elmer. The following oligonucleotides were used toprime the PCR: 5EF1αPolyA:

5' GAATTCTTTTTTGCGTGTGGCAG 3' (SEQ ID NO:78) and 3EFl1αPolyA:

5' ATCGATATTCCTTCCCCTTCC 3' (SEQ ID NO:79). The 3EF1αPolyA

oligonucleotide generates a ClaI site at the 3' end of the poly A siteand the 5EF1αPolyA oligonucleotide generates an EcoRI site at the 5' endof the poly A site. Digestion of the PCR product with EcoRI and ClaIyields a 292 bp EcoRI/ClaI fragment.

pSSD5 (Ex. 1) was digested with PvuII and a ClaI linkers (NEB,unphosphorylated) were ligated to the PvuII ends to convert the PvuIIsite located at the 3' end of the SV40 poly A site to a ClaI site. Theresulting construct was then digested with SalI and ClaI and the ˜2.1 kbfragment containing the plasmid backbone (e.g., the Amp^(R) gene andplasmid ORI) was isolated and ligated to an ˜870 bp SalI/EcoRI fragmentcontaining the Moloney enhancer/promoter, splice donor/acceptor andpolylinker isolated from pMSD5 (Ex. 1) together with the 292 bpEcoRI/ClaI fragment containing the poly A site of the human elongationfactor 1α gene to generate pMSD8.

b) Collection of Tumor Cells

Cells are collected by either surgical biopsy of enlarged lymph nodes orby fine needle biopsy of effected lymph nodes. The biopsy sample israpidly frozen on dry ice.

c) Isolation of RNA From Tumor Cells

RNA is isolated from the biopsy sample by using a variety of standardtechniques or commercially available kits. For example, kits which allowthe isolation of RNA from tissue samples are available from Qiagen, Inc.(Chatsworth, Calif.) and Stratagene (LaJolla, Calif.), respectively.Total RNA may be isolated from tissues and tumors by a number of methodsknown to those skilled in the art and commercial kits are available tofacilitate the isolation. For example, the RNeasy® kit (Qiagen Inc.,Chatsworth, Calif.) provides protocol, reagents and plasticware topermit the isolation of total RNA from tissues, cultured cells orbacteria, with no modification to the manufacturer's instructions, inapproximately 20 minutes. Should it be desirable to further enrich formessenger RNAs, the polyadenylated RNAs in the mixture may bespecifically isolated by binding to an oligo-deoxythymidine matrix,through the use of a kit such as the Oligotex® kit (Qiagen). Comparableisolation kits for both of these steps are available through a number ofcommercial suppliers.

In addition, RNA may be extracted from samples, including biopsyspecimens, conveniently by lysing the homogenized tissue in a buffercontaining 0.22 M NaCl, 0.75 mM MgCl₂, 0.1 M Tris-HCl, pH 8.0, 12.5 mMEDTA, 0.25% NP40, 1% SDS, 0.5 mM DTT, 500 u/ml placental RNAse inhibitorand 200 μg/ml Proteinase K. Following incubation at 37° C. for 30 min,the RNA is extracted with phenol:chloroform (1:1) and the RNA isrecovered by ethanol precipitation.

A particularly preferred method for the isolation of total cellular RNAfrom patient tumor samples is the RNAzol method (Teltest, Inc.,Friendswood, Tex.) which is performed according to the manufacturer'sinstructions.

d) Cloning of Ig Genes from Tumor Cells

Because the first and third complementarity determining regions (CDRs)of rearranged immunoglobulin genes are flanked by conserved sequences,it is possible to design PCR primers capable of amplifying cDNA for thevariable regions from mRNA derived from Ig-expressing tumor cellswithout any specific knowledge of the nucleotide sequence of thatspecific antibody. Primers suitable for isolating the variable regionsfrom a patient's tumor are provided below.

Using total cellular RNA isolated from the tumor, double stranded (ds)cDNA is generated as described in Example 3 with the exception that 20μg of total cellular RNA is used instead of poly A⁺ RNA. Five percent ofthe ds cDNA preparation is used for each PCR reaction. [Alternatively,ds cDNA may be produced using the technique of RT-PCR (reversetranscription-PCR); kits which permit the user to start with tissue andproduce a PCR product are available from Perkin Elmer (Norwalk Conn.)and Stratagene (LaJolla, Calif.). The RT-PCR technique generates asingle-stranded cDNA corresponding to a chosen segment of the codingregion of a gene by using reverse transcription of RNA; thesingle-stranded cDNA is then used as template in the PCR].

PCR reactions are carried out in a final volume of 50 μl and contain 1×Pfu Buffer (Stratagene), all 4 dNTPs at 100 μM each, primers at 0.5 μMeach, Pfu polymerase (Stratagene) and 5% of the ds cDNA preparation. Thereactions are thermocycled as follows: 94° C., 15 sec; 60° C., 30 see;75° C., 1.5 min for 15-30 cycles. Aliquots (5 μl) are removed after 15,20, 25 and 30 cycles to examine the appearance of the primary PCRproduct. Preparative reactions of 200 μl using the correct V regionprimers will be then run for cloning purposes.

Prior to conducting a PCR reaction to obtain Ig sequences from apatient's tumor, the tumor is immunophenotyped using commerciallyavailable antibodies to determine the heavy chain and light chainisotypes; this allows the number of PCRs to be minimized. For example,if the Ig expressed by the patient's tumor utilizes a μ heavy chain anda κ light chain, then PCR reactions described below which contain Cγ andCλ primers need not be run. However, the use of PCR primerscorresponding to heavy and light chain isotypes which are not utilized(according to the immunophenotyping results) by the patient's tumorserves as a convenient means to confirm the immunophenotyping results.

PCR primers utilized to clone variable regions of the patient'stumor-specific Ig are summarized below in Tables 1 through 3:

                                      TABLE 1                                     __________________________________________________________________________    Heavy Chain Primers                                                           __________________________________________________________________________    VH1L                                                                              5'40 -TCT AGA ATT CAC GCG TCC ACC ATG GAC TGG ACC TGG AG-3'40                                                        SEQ ID NO:52                          - VH2L                  5'40 -TCT AGA ATT CAC GCG TCC ACC ATG GAC ACA                                                 CTT TGC TAC AC -3'40                                                                       SEQ ID NO:53                                                       - VH3L                 5'40                                                 -TCT AGA ATT CAC GCG TCC ACC                                                  ATG GAG TTT GGG CTG AGC                                                       TGG-3'40                                                                       SEQ ID NO:54                         - VH4L               5'40 -TCT AGA ATT CAC GCG TCC ACC ATG AAA CAC CTG                                                TGG TTC TTC CT-3'40                                                                   SEQ ID NO:55                  - VH5L               5'40 -TCT AGA ATT CAC GCG TCC ACC ATG GGG TCA ACC                                                GCC ATC CT-3'40                                                                      SEQ ID NO:56                   - VH6L              5'40 -TCT AGA ATT CAC GCG TCC ACC ATG TCT GTC TCC                                                 TTC CTC ATC TT-3'40                                                                      SEQ ID NO:57                                                         - C.sub.γ                                                                 5'40 -GCC TGA GTT CCA CGA                                                 CAC CGT CAC-3'40         SEQ                                                  ID NO:58                              - C.sub.μ                 5'40 -GGG GAA AAG GGT TGG GGC GGA                                                        TGC-3'40         SEQ ID NO:59                                                   - JH1245               5'40                                                 -GAG GGG CCC TTG GTC GAC GCT                                                  GAG GAG ACG GTG ACC AGG-3                                                                        SEQ ID                                                     NO:60                                 - JH3            5'40 -GAG GGG CCC TTG GTC GAC GCT GAA GAG ACG GTG ACC                                                ATT G -3'40                                                                      SEQ ID NO:61                       - JH6       5'40 -GAG GGG CCC TTG GTC GAC GCT GAG GAG ACG GTG ACC                                                     GTG-3'40                                                                       SEQ ID NO:62                      __________________________________________________________________________

                                      TABLE 2                                     __________________________________________________________________________    Kappa Chain Primers:                                                          __________________________________________________________________________    V.sub.κ I                                                                   5'40 -TCT AGA ATT CAC GCG TCC ACC ATGGAC ATG AGG GTC CCC GCT CAG-3'40                                                SEQ ID NO:63                          - V.sub.κ II    5'40 -TCT AGA ATT CAC GCG TCC ACC ATG AGG CTC                                                   CCT GCT CAG C-3'40                                                                           SEQ ID NO:64                                                     - V.sub.κ III                                                           5'40 -TCT AGA ATT CAC GCG                                                   TCC ACC ATG GAA GCC CCA GCG                                                   CAG CTT-3'40                                                                        SEQ ID NO:65                    - V.sub.κ IV    5'40 -TCT AGA ATT CAC GCG TCC ACC ATG GTG TTG                                                   CAG ACC CAG GT-3'40                                                                           SEQ ID NO:66                                                    - V.sub.κ V                                                            5'40 -TCT AGA ATT CAC GCG TCC                                                ACC ATG GGG TCC CAG GTT CAC                                                   CT-3'40                                                                           SEQ ID NO:67                      - V.sub.κ VIa             5'40 -TCT AGA ATT CAC GCG TCC ACC ATG                                                 TTG CCA TCA CAA CTC ATT G-3'40                                                                         SEQ                                                  ID NO:68                              - V.sub.κ VIb             5'40 -TCT AGA ATT CAC GCG TCC ACC ATG                                                 GTG TCC CCGTTG CAA TT-3'40                                                                           SEQ ID                                                 NO:69                                 - C.sub.κ     5'40 -GGT TCC GGA CTT AAG CTG CTC ATC AGA TGG CGG                                                 G-3'40   SEQ ID NO:70              __________________________________________________________________________

                                      TABLE 3                                     __________________________________________________________________________    Lambda Chain Primers:                                                         __________________________________________________________________________    VL1                                                                             5'40 -TCT AGA ATT CAC GCG TCC ACC ATG GCC TGCTCT CCT CTC CTC CT-3'40                                                  SEQ ID NO:71                           - VL2         5'40 -TCT AGA ATT CAC GCG TCC ACC ATG GCC TGG GCT CTG                                                  CTG CTC CT-3'40                                                                        SEQ ID NO:72                  - VL3         5'40 -TCT AGA ATT CAC GCG TCC ACC ATG GCC TGG ATC CTT                                                  CTC CTC CTC-3'40                                                                       SEQ ID NO:73                  - VL4         5'40 -TCT AGA ATT CAC GCG TCC ACC ATG GCC TGG ACC CCT                                                  CTC TGG CTC-3'40                                                                       SEQ ID NO:74                  - VL6   5'40 -TCT AGA ATT CAC GCG TCC ACC ATG GCC TGG GCC CCA CTA                                                    CT-3'40                                                                          SEQ ID NO:75                        - VL8          5'40 -TCT AGA ATT CAC GCG TCC ACC ATG GCC TGG ATG ATG                                                 CTT CTC CT-3'40                                                                     SEQ ID NO:76                     - C.sub.   5'40 -GGC GCC GCC TTG GGC TGA CCT AGG ACG GT-3'40                                                         SEQ ID NO:77                        __________________________________________________________________________

The VH1-6L primers contain recognition sites for XbaI, EcoRI and MluI attheir 5' ends. The three JH primers contain recognition sites for ApaIand SalI at their 5' ends. The seven Vκ primers contain recognitionsites for XbaI, EcoRI and MluI at their 5' ends. The Cκ primer containsrecognition sites for BspEI and AflII at the 5' end. The six VL primerscontain recognition sites for XbaI, EcoRI and MluI at their 5' ends. TheCλ primer contains recognition sites for KasI and AvrII at the 5' end.

For each tumor sample, five V_(H) PCR reactions are run. Each V_(H)reaction will contain the Cμ and Cγ primers. The Cμ primer (SEQ IDNO:59) should result in ˜590 bp product for the heavy chain V (V_(H))region expressed in an IgM positive tumor. The Cγ primer (SEQ ID NO:58)should result in ˜480 bp product for the heavy chain V region expressedin an IgG positive tumor. The VH1, VH2, VH3, and VH4 primers (SEQ IDNOS: 52-55, respectively) are used in separate PCR reactions and the VH5and VH6 primers (SEQ ID NOS:56 and 57, respectively) are used togetherin the same reaction. The V_(H) primer(s), which when used in connectionwith a C_(H) region primer, gives a PCR product of the expected size isthen be used in three separate PCR reactions containing either theJH1245, JH3 or JH6 primers (SEQ ID NOS:60-62, respectively) to generatea PCR product corresponding to the variable (V), diversity (D) andjoining (J) regions present in the Ig(s) expressed by the patient'stumor. The VDJ reaction product is then subcloned into the pSRαSD9CG3Cvector or pSRαSD9CG4C vector using the 5' XbaI, EcoRI or MluI sites andthe 3' SalI or ApaI sites to provide an expression vector encoding thepatient's heavy chain variable domain linked to either a γ3 or γ4constant domain. As is understood by those in the art, the PCR productis subcloned into the expression vector using restriction enzymes whichlack sites internal to the PCR product (i.e., within the Ig sequences).The PCR products are digested with restriction enzymes that have siteslocated within the PCR primers to confirm that the PCR product lacks aninternal site for a given restriction enzyme prior to subcloning the PCRproduct into the desired expression vector. It is anticipated that the5' MluI site can be employed for each PCR product given that MluI sitesare very infrequently found in the genome; however the 5' primers alsocontain XbaI and EcoRI sites in the event a particular PCR productcontains an internal MluI site. The following restriction enzymes (whichhave recognition sites in the above-described 3' PCR primers) areexamined first for their inability to cut internally to the PCRproducts: SalI for heavy chain PCR products; AflII for kappa light chainPCR products; AvrII for lambda light chain PCR products. As discussedabove, each 3' PCR primer provides alternative restriction enzyme sites.

With regard to choosing an expression vector, the pSRαSD9CG3C vector isinitially chosen as Cγ3 is the least frequently used isotype in humans(Cγ4 is the next least frequently utilized isotype, with Cγ1 and Cγ2being the most frequently used isotypes) and therefore ELISAs performedfollowing immunization with a vaccine comprising Cγ3 are easier toconduct and interpret as the patient's anti-idiotype response willmainly consist of the yl and y2 isotypes. However, Cγ4 may be chosenover Cγ3 if a given Cγ3 construct produces an Ig protein which tends tofall out of solution upon purification.

For each tumor sample, five Vκ PCR reactions are run. Each Vκ PCRreaction will contain the Cκ primer (SEQ ID NO:70). The VκI, VκII, andVκIII primers (SEQ ID NOS:63-65, respectively) will be run in separatereactions. The VκIV and VκV primers (SEQ ID NOS:66 and 67, respectively)are combined in one PCR reaction and the VκVIa and VκVIb primers (SEQ IDNOS:68 and 69, respectively) in another. The PCR reaction which yields aPCR product of the expected size (˜480 bp) is used as the source of DNAencoding the variable domain derived from the light chain of thepatient's Ig. The positive reaction product is subcloned into thepSRαSD9CKC vector using the 5' XbaI, EcoRI or MluI sites and the 3'AflII or BspEI sites.

For each tumor sample, six Vλ PCR reactions are run. Each Vκ PCRreaction will contain the Cλ primer (SEQ ID NO:77). The VL1, VL2, VL3,VL4, VL6 and VL8 primers (SEQ ID NOS:71-76, respectively) are used inseparate reactions. The PCR reaction which yields a PCR product of theexpected size (˜420 bp) is used as the source of DNA encoding thevariable domain derived from the light chain of the patient's Ig. Thepositive reaction product will be subcloned into the pSRαSD9CL2C vectorusing the 5' XbaI, EcoRI or MluI sites and the 3' AvrII or KasI sites.It is understood by those skilled in the art that the tumor cells willexpress either a κ or a λ light chain. Therefore, it is expected that aPCR product will be recovered from either the Vκ or Vλ PCRs but not fromboth.

e) Expression and Amplification of Tumor-Specific Ig in Mammalian Cells

Once expression vectors containing sequences derived from the variableregions of the heavy and light chains found in the patient's tumor areconstructed, these plasmids are used to transform E. coli usingconventional techniques. Between 18 and 24 colonies from each subcloningare screened for heavy and light chain inserts as appropriate byrestriction enzyme analysis of miniprep DNA (from 1-1.5 ml cultures).Equal aliquots of the positive subclones are used to inoculate largercultures (˜250 mls) from which the DNA for electroporation is prepared.This allows for the isolation of the somatic variants in the tumorpopulation and result in transfectants (e.g., BW5147.G.1.4transfectants) expressing these somatic variants.

To further define the presence of somatic variants, 20 μl PCR reactionsare run using ˜100 pg of each miniprep DNA and the appropriate V regionand C region primers. Digestion of the resulting PCR products withseveral four base recognition restriction enzymes allows thedifferentiation of somatic variants. In addition, DNA sequencing can beperformed on individual subclones to demonstrate the presence of somaticvariants within the pool of subclones containing the cloned heavy andlight chain variable regions.

Plasmids encoding the chimeric heavy and light chains derived from thepatient's Ig are electroporated along with pM-HPRT-SSD9-DHER intoBW5147.G.1.4 cells as follows. The Ig expression plasmids (whichcomprise a mixture of vectors containing the somatic variants foundwithin the tumor Ig) are linearized by digestion with AscI or PacI.pM-HPRT-SSD9-DHFR is linearized with AscI or PacI. pM-HPRT-SSD9-DHFR andthe Ig expression plasmids are used at a ratio of 1:20-50. Approximately15 μg of pM-HPRT-SSD9-DHFR (10-20 μg) is used while a total of ˜500 μgof the expression vectors are used. The linearized plasmids aredigested, precipitated and resuspended in 0.5 ml electroporation buffer[i.e., 1× HBS(EP)] as described in Example 7. The linearized plasmids in0.5 ml electroporation buffer are mixed with 2×10⁷ cells (e.g.,BW5147.G.1.4) in 0.5 ml electroporation buffer and electroporated asdescribed in Example 7. The cells are then grown in selective mediumfollowed by growth in medium containing MTX as described in Examples 7and 8. Clones which grow in the selective medium are checked for theability to express the cloned Ig proteins using standard methods (e.g.,by ELISA). Primary selectants expressing high levels of the cloned Igproteins are then grown in medium containing MTX as described inExamples 7 and 8 to amplify the transfected genes. The presence of theselectable and amplifiable markers on a single piece of DNA (i.e.,pM-HPRT-SSD9-DHFR), obviates concerns that primary transfectants (i.e.,cells capable of growing in medium containing Hx and Az) which expressthe genes of interest (i.e., the Ig proteins) at high levels have failedto integrate a DHFR gene.

f) Purification of Tumor-Specific Ig From Amplified Cell Lines

The tumor-specific Ig expressed by the amplified cell lines (usingeither the pSRαSD9CG3C or pSRαSD9CG4C vectors) is purified bychromatography of culture supernatants on Protein G Sepharose(Pharmacia); Protein G binds to both IgG₃ and IgG₄. The chromatographyis conducted according to the manufacturer's instructions. When thetumor-specific Ig is produced using the pSRαSD9CG4C vector, Protein ASepharose (Pharmacia) may also be employed for purification.

g) Administration of Tumor-Specific Ig (Multivalent Vaccine)

The purified tumor immunoglobulin-idiotype protein may be conjugated toa protein carrier such as keyhole limpet hemocyanin (KLH) (Calbiochem,San Diego, Calif.) prior to administration to the patient. If theimmunoglobulin-idiotype protein is to be conjugated with KLH, the KLH isdepleted of endotoxin using methods known to the art [Kwak et al.(1992), supra]. For example, the KLH is applied to a QAE Zeta Prep 15disk (LKB, Broma, Sweeden) to produce a preparation of KLH containingless than 1000 endotoxin units per milliliter. Equal volumes of filtersterilized purified KLH and purified immunoglobulin-idiotype protein(each at 1 mg/ml) are mixed together. Sterile glutaraldehyde is added ata final concentration of 0.1%. The Ig-KLH conjugate is then dialyzedextensively against physiologic saline to remove excess glutaraldehyde.

Purified immunoglobulin-idiotype protein (conjugated or unconjugated) ismixed with an immunologic adjuvant such as SAF-1 (Syntex adjuvantformulation 1; Roche) or other adjuvant presently or subsequentlyapproved for administration to humans [e.g., QS-21 (Perlmmune, Inc.,Rockville, Md.)]. The purified immunoglobulin-idiotype protein isemulsified in the desired adjuvant and injected subcutaneously at 0, 2,6, 10 and 14 weeks. Booster injects may be given at 24 and 28 weeks.Each injection contains 0.5 mg of purified, tumor-specific idiotypeimmunoglobulin (which may be conjugated 1:1 with KLH).

An alternative to the use of KLH as a foreign carrier protein to boostthe immune response to the immunoglobulin idiotype protein is the use ofa fusion protein comprising idiotype protein and a cytokine (e.g.,GM-CSF, IL-2 or IL-4) [PCT International Application PCT/US93/09895,Publication No. WO 94/08601 and Tao and Levy (1993) Nature 362:755 andChen et al. (1994) J. Immunol. 153:4775]. In these fusion proteins,sequences encoding the desired cytokine are added to the 3' end ofsequences encoding the immunoglobulin idiotype protein. The presentinvention contemplates the use of idiotype-cytokine fusion proteins forthe treatment of B-cell lymphoma. The sequences encoding the heavy chainof the patient's immunoglobulin protein are cloned as described aboveand inserted into an expression vector containing sequences encoding thedesired cytokine such that a fusion protein comprising, from amino- tocarboxy-terminus, the heavy chain of the patient's tumor-specificimmunoglobulin and the desired cytokine.

An alternative to the use of foreign carrier proteins, cytokines, orimmunologic adjuvants is the use of autologous dendritic cells pulsedwith the purified immunoglobulin-idiotype protein [see for example, Hsuet al. (1996), supra and PCT International Application PCT/US91/01683,Publication No. WO 91/13632]. Methods for the isolation of humandendritic cells from peripheral blood are known to the art [Mehta et al.(1994) J. Immunol. 153:996 and Takamizawa et al. (1995) J. Clin. Invest.95:296]. Briefly, the patient is leukapheresed using a cell separator(COBE). Peripheral blood mononuclear cells (PBMCs) are collected byseparation through Ficoll-Hypaque (Pharmacia). Monocytes are thenremoved by centrifugation through discontinuous Percoll (Pharmacia)gradients. The monocyte-depleted PBMCs are then placed in medium (RPMI1640 containing 10% autologous patient serum) containing idiotypeprotein (2 μg/ml). Following incubation for 24 hours at 37° C. in ahumidified atmosphere containing 10% CO₂, the dendritic cells areseparated from lymphocytes by sequential centrifugation through 15% and14% (wt/vol) metrizamide gradients. The preparation is then incubatedfor 14-18 hours in medium containing 50 μg/ml idiotype protein. Thecells are then washed to remove free antigen (i.e., idiotype protein)and placed in sterile saline containing 5% autologous serum andadministered intravenously.

Each patient is followed to determine the production ofidiotype-specific antibody; the in vitro proliferative responses (toKLH, if used, and to immunoglobulin idiotype using 0 to 100 μg ofsoluble protein per milliliter in 5 day in vitro cultures) of PBMCsisolated from the treated patients may also be determined. These assaysare conducted immediately before each immunization and 1 to 2 monthsfollowing the last immunization. Patients are monitored for diseaseactivity by physical examination, routine laboratory studies and routineradiographic studies. Regression of lymph nodes or cutaneouslymphomatous masses may be confirmed by computed tomography (CT). Inaddition, residual disease may be measured using a tumor-specific CDR3analysis as described by Hsu et al. (1996), supra.

h) Treatment of T-cell Tumors

Vaccines comprising soluble T cell receptor (TCR) proteins derived froma patient's T cell tumor (i.e., a T cell leukemia or lymphoma) areproduced using the methods described in Example 9 with the exceptionthat pM-HPRT-SSD9-DHFR is used in place of separate selection andamplification vectors as described above. The thrombin solubilized TCRproteins are purified by chromatography on a resin comprising amonoclonal antibody (mcab) directed against a monomorphic determinant onhuman αβ TCRs [e.g., mcab T10B9.1A-31 (Pharmingen, San Diego, Calif.);mcab BMA031 (Immunotech, Westbrook, Me.); mcabs BW242/412, 8A3 or 3A8(Serotec, Washington, DC). Antibodies directed against monomorphic(i.e., invariant) determinants on TCRs recognize all αβ TCRs.

The purified tumor-specific idiotype TCR protein is administered asdescribed above for the purified tumor-specific idiotype Ig protein(i.e., mixing with an immunologic adjuvant, conjugation to a proteincarrier, the use of TCR-cytokine fusion proteins, the use of dendriticcells pulsed with the purified TCR protein such as SAF-1, etc.).Patients are followed to determine the production of idiotype-specificantibody as described above. Patients are monitored for disease activityby physical examination, routine laboratory studies and routineradiographic studies.

From the above, it is clear that the present invention provides improvedmethods for the amplification and expression of recombinant genes incells. The resulting amplified cell lines provide large quantities ofrecombinant proteins in a short period of time. The ability to producelarge quantities of recombinant proteins in a short period of time isparticularly advantageous when proteins unique to a patient's tumors areto be used for therapeutic purposes, such as for vaccination.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 80                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - TCTAGAGCGG CCGCGGAGGC CGAATTCG         - #                  - #                 28                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - GATCCGAATT CGGCCTCCGC GGCCGCTCTA GATGCA      - #                  -     #       36                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 677 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - GGATCCAGAC ATGATAAGAT ACATTGATGA GTTTGGACAA ACCACAACTA GA -             #ATGCAGTG     60                                                                 - - AAAAAAATGC TTTATTTGTG AAATTTGTGA TGCTATTGCT TTATTTGTAA CC -            #ATTATAAG    120                                                                 - - CTGCAATAAA CAAGTTAACA ACAACAATTG CATTCATTTT ATGTTTCAGG TT -            #CAGGGGGA    180                                                                 - - GGTGTGGGAG GTTTTTTAAA GCAAGTAAAA CCTCTACAAA TGTGGTATGG CT -            #GATTATGA    240                                                                 - - TCATGAACAG ACTGTGAGGA CTGAGGGGCC TGAAATGAGC CTTGGGACTG TG -            #AATCAATG    300                                                                 - - CCTGTTTCAT GCCCTGAGTC TTCCATGTTC TTCTCCCCAC CATCTTCATT TT -            #TATCAGCA    360                                                                 - - TTTTCCTGGC TGTCTTCATC ATCATCATCA CTGTTTCTTA GCCAATCTAA AA -            #CTCCAATT    420                                                                 - - CCCATAGCCA CATTAAACTT CATTTTTTGA TACACTGACA AACTAAACTC TT -            #TGTCCAAT    480                                                                 - - CTCTCTTTCC ACTCCACAAT TCTGCTCTGA ATACTTTGAG CAAACTCAGC CA -            #CAGGTCTG    540                                                                 - - TACCAAATTA ACATAAGAAG CAAAGCAATG CCACTTTGAA TTATTCTCTT TT -            #CTAACAAA    600                                                                 - - AACTCACTGC GTTCCAGGCA ATGCTTTAAA TAATCTTTGG GCCTAAAATC TA -            #TTTGTTTT    660                                                                 - - ACAAATCTGG CCTGCAG             - #                  - #                      - #  677                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - CTAGAATTCA CGCGTAGGCC TCCGCGGCCG CGCGCATGC      - #                      - #    39                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - AATTGCATGC GCGCGGCCGC GGAGGCCTAC GCGTGAATT      - #                      - #    39                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 633 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - CAAGCTTGCT GTGGAATGTG TGTCAGTTAG GGTGTGGAAA GTCCCCAGGC TC -             #CCCAGCAG     60                                                                 - - GCAGAAGTAT GCAAAGCATG CATCTCAATT AGTCAGCAAC CAGGTGTGGA AA -            #GTCCCCAG    120                                                                 - - GCTCCCCAGC AGGCAGAAGT ATGCAAAGCA TGCATCTCAA TTAGTCAGCA AC -            #CATAGTCC    180                                                                 - - CGCCCCTAAC TCCGCCCATC CCGCCCCTAA CTCCGCCCAG TTCCGCCCAT TC -            #TCCGCCCC    240                                                                 - - ATGGCTGACT AATTTTTTTT ATTTATGCAG AGGCCGAGGC CGCCTCGGCC TC -            #TGAGCTAT    300                                                                 - - TCCAGAAGTA GTGAGGAGGC TTTTTTGGAG GCCTAGGCTT TTGCAAAAAG CT -            #CCTCGAGC    360                                                                 - - TCGCATCTCT CCTTCACGCG CCCGCCGCCC TACCTGAGGC CGCCATCCAC GC -            #CGGTTGAG    420                                                                 - - TCGCGTTCTG CCGCCTCCCG CCTGTGGTGC CTCCTGAACT GCGTCCGCCG TC -            #TAGGTAAG    480                                                                 - - TTTAGAGCTC AGGTCGAGAC CGGGCCTTTG TCCGGCGCTC CCTTGGAGCC TA -            #CCTAGACT    540                                                                 - - CAGCCGGCTC TCCACGCTTT GCCTGACCCT GCTTGCTCAA CTCTACGTCT TT -            #GTTTCGTT    600                                                                 - - TTCTGTTCTG CGCCGTTACA GATCGCCTCG AGG       - #                  -      #        633                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 635 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - CAAGCTTGCG ATTAGTCCAA TTTGTTAAAG ACAGGATATC AGTGGTCCAG GC -            #TCTAGTTT     60                                                                 - - TGACTCAACA ATATCACCAG CTGAAGCCTA TAGAGTACGA GCCATAGATA AA -            #ATAAAAGA    120                                                                 - - TTTTATTTAG TCTCCAGAAA AAGGGGGGAA TGAAAGACCC CACCTGTAGG TT -            #TGGCAAGC    180                                                                 - - TAGCTTAAGT AACGCCATTT TGCAAGGCAT GGAAAAATAC ATAACTGAGA AT -            #AGAGAAGT    240                                                                 - - TCAGATCAAG GTCAGGAACA GATGGAACAG CTGAATATGG GCCAAACAGG AT -            #ATCTGTGG    300                                                                 - - TAAGCAGTTC CTGCCCCGGC TCAGGGCCAA GAACAGATGG AACAGCTGAA TA -            #TGGGCCAA    360                                                                 - - ACAGGATATC TGTGGTAAGC AGTTCCTGCC CCGGCTCAGG GCCAAGAACA GA -            #TGGTCCCC    420                                                                 - - AGATGCGGTC CAGCCCTCAG CAGTTTCTAG AGAACCATCA GATGTTTCCA GG -            #GTGCCCCA    480                                                                 - - AGGACCTGAA ATGACCCTGT GCCTTATTTG AACTAACCAA TCAGTTCGCT TC -            #TCGCTTCT    540                                                                 - - GTTCGCGCGC TTCTGCTCCC CGAGCTCAAT AAAAGAGCCC ACAACCCCTC AC -            #TCGGGGCG    600                                                                 - - CCAGTCCTCC GATTGACTGA GTCGCCCCCT CGAGG       - #                       - #      635                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 483 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - AAGCTTTGGA GCTAAGCCAG CAATGGTAGA GGGAAGATTC TGCACGTCCC TT -            #CCAGGCGG     60                                                                 - - CCTCCCCGTC ACCACCCCCC CCAACCCGCC CCGACCGGAG CTGAGAGTAA TT -            #CATACAAA    120                                                                 - - AGGACTCGCC CCTGCCTTGG GGAATCCCAG GGACCGTCGT TAAACTCCCA CT -            #AACGTAGA    180                                                                 - - ACCCAGAGAT CGCTGCGTTC CCGCCCCCTC ACCCGCCCGC TCTCGTCATC AC -            #TGAGGTGG    240                                                                 - - AGAAGAGCAT GCGTGAGGCT CCGGTGCCCG TCAGTGGGCA GAGCGCACAT CG -            #CCCACAGT    300                                                                 - - CCCCGAGAAG TTGGGGGGAG GGGTCGGCAA TTGAACCGGT GCCTAGAGAA GG -            #TGGCGCGG    360                                                                 - - GGTAAACTGG GAAAGTGATG TCGTGTACTG GCTCCGCCTT TTTCCCGAGG GT -            #GGGGGAGA    420                                                                 - - ACCGTATATA AGTGCAGTAG TCGCCGTGAA CGTTCTTTTT CGCAACGGGT TT -            #GCCGCCTC    480                                                                 - - GAG                  - #                  - #                  - #                483                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - AAGCTTTGGA GCTAAGCCAG CAAT          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - CTCGAGGCGG CAAACCCGTT GCG           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1451 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - AAGCTTTGGA GCTAAGCCAG CAATGGTAGA GGGAAGATTC TGCACGTCCC TT -             #CCAGGCGG     60                                                                 - - CCTCCCCGTC ACCACCCCCC CCAACCCGCC CCGACCGGAG CTGAGAGTAA TT -            #CATACAAA    120                                                                 - - AGGACTCGCC CCTGCCTTGG GGAATCCCAG GGACCGTCGT TAAACTCCCA CT -            #AACGTAGA    180                                                                 - - ACCCAGAGAT CGCTGCGTTC CCGCCCCCTC ACCCGCCCGC TCTCGTCATC AC -            #TGAGGTGG    240                                                                 - - AGAAGAGCCA TGCGTGAGGC TCCGGTGCCC GTCAGTGGGC AGAGCGCACA TC -            #GCCCACAG    300                                                                 - - TCCCCGAGAA GTTGGGGGGA GGGGTCGGCA ATTGAACCGG TGCCTAGAGA AG -            #GTGGCGCG    360                                                                 - - GGGTAAACTG GGAAAGTGAT GTCGTGTACT GGCTCCGCCT TTTTCCCGAG GG -            #TGGGGGAG    420                                                                 - - AACCCGTATA TAAGTGCAGT AGTCGCCGTG AACGTTCTTT TTCGCAACGG GT -            #TTGCCGCC    480                                                                 - - AGAACACAGG TAAGTGCCGT GTGTGGTTCC CGCGGGCCTG GCCTCTTTAC GG -            #GTTATGGC    540                                                                 - - CCTTGCGTGC CTTGAATTAC TTCCACGCCC CTGGCTGCAG TACGTGATTC TT -            #GATCCCGA    600                                                                 - - GCTTCGGGTT GGAAGTGGGT GGGAGAGTTC GAGGCCTTGC GCTTAAGGAG CC -            #CCTTCGCC    660                                                                 - - TCGTGCTTGA GTTGAGGCCT GGCCTGGGCG CTGGGGCCCC CGCGTGCGAA TC -            #TGGTGGCA    720                                                                 - - CCTTCGCGCC TGTCTCGCTG CTTTCGATAA GTCTCTAGCC ATTTAAAATT TT -            #TGATGACC    780                                                                 - - TGCTGCGACG CTTTTTTTCT GGCAAGATAG TCTTGTAAAT GCGGGCCAAG AT -            #CTGCACAC    840                                                                 - - TGGTATTTCG GTTTTTGGGG CCGCGGGCGG CGACGGGGCC CGTGCGTCCC AG -            #CGCACATG    900                                                                 - - TTCGGCGAGG CGGGGCCTGC GAGCGCGGCC ACCGAGAATC GGACGGGGGT AG -            #TCTCAAGC    960                                                                 - - TGGCCGGCCT GCTCTGGTGC CTGGCCTCGC GCCGCCGTGT ATCGCCCCGC CC -            #TGGGCGGC   1020                                                                 - - AAGGCTGGCC CGGTCGGCAC CAGTTGCGTG AGCGGAAAGA TGGCCGCTTC CC -            #GGCCCTGC   1080                                                                 - - TGCAGGGAGC TCAAAATGGA GGACGCGGCG CTCGGGAGAG CGGGCGGGTG AG -            #TCACCCAC   1140                                                                 - - ACAAAGGAAA AGGGCCTTTC CGTCCTCAGC CGTCGCTTCA TGTGACTCCA CG -            #GAGTACCG   1200                                                                 - - GGCGCCGTCC AGGCACCTCG ATTAGTTCTC GAGCTTTTGG AGTACGTCGT CT -            #TTAGGTTG   1260                                                                 - - GGGGGAGGGG TTTTATGCGA TGGAGTTTCC CCACACTGAG TGGGTGGAGA CT -            #GAAGTTAG   1320                                                                 - - GCCAGCTTGG CACTTGATGT AATTCTCCTT GGAATTTGCC CTTTTTGAGT TT -            #GGATCTTG   1380                                                                 - - GTTCATTCTC AAGCCTCAGA CAGTGGTTCA AAGTTTTTTT CTTCCATTTC AG -            #GTGTCGTG   1440                                                                 - - AAAACTCTAG A               - #                  - #                      - #     1451                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - TCTAGAGTTT TCACGACACC TGA           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1289 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 88..741                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - TTACCTCACT GCTTTCCGGA GCGGTAGCAC CTCCTCCGCC GGCTTCCTCC TC -             #AGACCGCT     60                                                                 - - TTTTGCCGCG AGCCGACCGG TCCCGTC ATG CCG ACC CGC AGT - #CCC AGC GTC            111                                                                                         - #            Met Pro Thr Ar - #g Ser Pro Ser Val                            - #              1    - #           5                        - - GTG ATT AGC GAT GAT GAA CCA GGT TAT GAC CT - #A GAT TTG TTT TGT ATA          159                                                                       Val Ile Ser Asp Asp Glu Pro Gly Tyr Asp Le - #u Asp Leu Phe Cys Ile                10             - #     15             - #     20                          - - CCT AAT CAT TAT GCC GAG GAT TTG GAA AAA GT - #G TTT ATT CCT CAT GGA          207                                                                       Pro Asn His Tyr Ala Glu Asp Leu Glu Lys Va - #l Phe Ile Pro His Gly            25                 - # 30                 - # 35                 - # 40       - - CTG ATT ATG GAC AGG ACT GAA AGA CTT GCT CG - #A GAT GTC ATG AAG GAG          255                                                                       Leu Ile Met Asp Arg Thr Glu Arg Leu Ala Ar - #g Asp Val Met Lys Glu                            45 - #                 50 - #                 55              - - ATG GGA GGC CAT CAC ATT GTG GCC CTC TGT GT - #G CTC AAG GGG GGC TAT          303                                                                       Met Gly Gly His His Ile Val Ala Leu Cys Va - #l Leu Lys Gly Gly Tyr                        60     - #             65     - #             70                  - - AAG TTC TTT GCT GAC CTG CTG GAT TAC ATT AA - #A GCA CTG AAT AGA AAT          351                                                                       Lys Phe Phe Ala Asp Leu Leu Asp Tyr Ile Ly - #s Ala Leu Asn Arg Asn                    75         - #         80         - #         85                      - - AGT GAT AGA TCC ATT CCT ATG ACT GTA GAT TT - #T ATC AGA CTG AAG AGC          399                                                                       Ser Asp Arg Ser Ile Pro Met Thr Val Asp Ph - #e Ile Arg Leu Lys Ser                90             - #     95             - #    100                          - - TAC TGT AAT GAT CAG TCA ACG GGG GAC ATA AA - #A GTT ATT GGT GGA GAT          447                                                                       Tyr Cys Asn Asp Gln Ser Thr Gly Asp Ile Ly - #s Val Ile Gly Gly Asp           105                 1 - #10                 1 - #15                 1 -      #20                                                                              - - GAT CTC TCA ACT TTA ACT GGA AAG AAT GTC TT - #G ATT GTT GAA GAT        ATA      495                                                                    Asp Leu Ser Thr Leu Thr Gly Lys Asn Val Le - #u Ile Val Glu Asp Ile                          125  - #               130  - #               135              - - ATT GAC ACT GGT AAA ACA ATG CAA ACT TTG CT - #T TCC CTG GTT AAG CAG          543                                                                       Ile Asp Thr Gly Lys Thr Met Gln Thr Leu Le - #u Ser Leu Val Lys Gln                       140      - #           145      - #           150                  - - TAC AGC CCC AAA ATG GTT AAG GTT GCA AGC TT - #G CTG GTG AAA AGG ACC          591                                                                       Tyr Ser Pro Lys Met Val Lys Val Ala Ser Le - #u Leu Val Lys Arg Thr                   155          - #       160          - #       165                      - - TCT CGA AGT GTT GGA TAC AGG CCA GAC TTT GT - #T GGA TTT GAA ATT CCA          639                                                                       Ser Arg Ser Val Gly Tyr Arg Pro Asp Phe Va - #l Gly Phe Glu Ile Pro               170              - #   175              - #   180                          - - GAC AAG TTT GTT GTT GGA TAT GCC CTT GAC TA - #T AAT GAG TAC TTC AGG          687                                                                       Asp Lys Phe Val Val Gly Tyr Ala Leu Asp Ty - #r Asn Glu Tyr Phe Arg           185                 1 - #90                 1 - #95                 2 -      #00                                                                              - - AAT TTG AAT CAC GTT TGT GTC ATT AGT GAA AC - #T GGA AAA GCC AAA        TAC      735                                                                    Asn Leu Asn His Val Cys Val Ile Ser Glu Th - #r Gly Lys Ala Lys Tyr                          205  - #               210  - #               215              - - AAA GCC TAAGATGAGC GCAAGTTGAA TCTGCAAATA CGAGGAGTCC TG - #TTGATGTT           791                                                                       Lys Ala                                                                        - - GCCAGTAAAA TTAGCAGGTG TTCTAGTCCT GTGGCCATCT GCCTAGTAAA GC -             #TTTTTGCA    851                                                                 - - TGAACCTTCT ATGAATGTTA CTGTTTTATT TTTAGAAATG TCAGTTGCTG CG -            #TCCCCAGA    911                                                                 - - CTTTTGATTT GCACTATGAG CCTATAGGCC AGCCTACCCT CTGGTAGATT GT -            #CGCTTATC    971                                                                 - - TTGTAAGAAA AACAAATCTC TTAAATTACC ACTTTTAAAT AATAATACTG AG -            #ATTGTATC   1031                                                                 - - TGTAAGAAGG ATTTAAAGAG AAGCTATATT AGTTTTTTAA TTGGTATTTT AA -            #TTTTTATA   1091                                                                 - - TATTCAGGAG AGAAAGATGT GATTGATATT GTTAATTTAG ACGAGTCTGA AG -            #CTCTCGAT   1151                                                                 - - TTCCTATCAG TAACAGCATC TAAGAGGTTT TGCTCAGTGG AATAAACATG TT -            #TCAGCAGT   1211                                                                 - - GTTGGCTGTA TTTTCCCACT TTCAGTAAAT CGTTGTCAAC AGTTCCTTTT AA -            #ATGCAAAT   1271                                                                 - - AAATAAATTC TAAAAATT             - #                  - #                      - #1289                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 218 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - Met Pro Thr Arg Ser Pro Ser Val Val Ile Se - #r Asp Asp Glu Pro Gly        1               5 - #                 10 - #                 15              - - Tyr Asp Leu Asp Leu Phe Cys Ile Pro Asn Hi - #s Tyr Ala Glu Asp Leu                   20     - #             25     - #             30                  - - Glu Lys Val Phe Ile Pro His Gly Leu Ile Me - #t Asp Arg Thr Glu Arg               35         - #         40         - #         45                      - - Leu Ala Arg Asp Val Met Lys Glu Met Gly Gl - #y His His Ile Val Ala           50             - #     55             - #     60                          - - Leu Cys Val Leu Lys Gly Gly Tyr Lys Phe Ph - #e Ala Asp Leu Leu Asp       65                 - # 70                 - # 75                 - # 80       - - Tyr Ile Lys Ala Leu Asn Arg Asn Ser Asp Ar - #g Ser Ile Pro Met Thr                       85 - #                 90 - #                 95              - - Val Asp Phe Ile Arg Leu Lys Ser Tyr Cys As - #n Asp Gln Ser Thr Gly                  100      - #           105      - #           110                  - - Asp Ile Lys Val Ile Gly Gly Asp Asp Leu Se - #r Thr Leu Thr Gly Lys              115          - #       120          - #       125                      - - Asn Val Leu Ile Val Glu Asp Ile Ile Asp Th - #r Gly Lys Thr Met Gln          130              - #   135              - #   140                          - - Thr Leu Leu Ser Leu Val Lys Gln Tyr Ser Pr - #o Lys Met Val Lys Val      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ala Ser Leu Leu Val Lys Arg Thr Ser Arg Se - #r Val Gly Tyr Arg        Pro                                                                                             165  - #               170  - #               175             - - Asp Phe Val Gly Phe Glu Ile Pro Asp Lys Ph - #e Val Val Gly Tyr Ala                  180      - #           185      - #           190                  - - Leu Asp Tyr Asn Glu Tyr Phe Arg Asn Leu As - #n His Val Cys Val Ile              195          - #       200          - #       205                      - - Ser Glu Thr Gly Lys Ala Lys Tyr Lys Ala                                      210              - #   215                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - GCATGCGCGC GGCCGCGGAG GCTTTTTTTT TTTTTTTTTT     - #                      - #    40                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - CGGCAACGCG TGCCATCATG GTTCGAC          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - CGGCAGCGGC CGCATAGATC TAAAGCCAGC         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 671 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 13..573                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - ACGCGTGCCA TC ATG GTT CGA CCA TTG AAC TGC ATC - # GTC GCC GTG TCC             48                                                                                     Met Val Ar - #g Pro Leu Asn Cys Ile Val Ala Val Ser                             1  - #             5     - #             10                    - - CAA AAT ATG GGG ATT GGC AAG AAC GGA GAC CT - #A CCC TGG CCT CCG CTC           96                                                                       Gln Asn Met Gly Ile Gly Lys Asn Gly Asp Le - #u Pro Trp Pro Pro Leu                    15         - #         20         - #         25                      - - AGG AAC GAG TTC AAG TAC TTC CAA AGA ATG AC - #C ACA ACC TCT TCA GTG          144                                                                       Arg Asn Glu Phe Lys Tyr Phe Gln Arg Met Th - #r Thr Thr Ser Ser Val                30             - #     35             - #     40                          - - GAA GGT AAA CAG AAT CTG GTG ATT ATG GGT AG - #G AAA ACC TGG TTC TCC          192                                                                       Glu Gly Lys Gln Asn Leu Val Ile Met Gly Ar - #g Lys Thr Trp Phe Ser            45                 - # 50                 - # 55                 - # 60       - - ATT CCT GAG AAG AAT CGA CCT TTA AAG GAC AG - #A ATT AAT ATA GTT CTC          240                                                                       Ile Pro Glu Lys Asn Arg Pro Leu Lys Asp Ar - #g Ile Asn Ile Val Leu                            65 - #                 70 - #                 75              - - AGT AGA GAA CTC AAA GAA CCA CCA CGA GGA GC - #T CAT TTT CTT GCC AAA          288                                                                       Ser Arg Glu Leu Lys Glu Pro Pro Arg Gly Al - #a His Phe Leu Ala Lys                        80     - #             85     - #             90                  - - AGT TTG GAT GAT GCC TTA AGA CTT ATT GAA CA - #A CCG GAA TTG GCA AGT          336                                                                       Ser Leu Asp Asp Ala Leu Arg Leu Ile Glu Gl - #n Pro Glu Leu Ala Ser                    95         - #        100         - #        105                      - - AAA GTA GAC ATG GTT TGG ATA GTC GGA GGC AG - #T TCT GTT TAC CAG GAA          384                                                                       Lys Val Asp Met Val Trp Ile Val Gly Gly Se - #r Ser Val Tyr Gln Glu               110              - #   115              - #   120                          - - GCC ATG AAT CAA CCA GGC CAC CTT AGA CTC TT - #T GTG ACA AGG ATC ATG          432                                                                       Ala Met Asn Gln Pro Gly His Leu Arg Leu Ph - #e Val Thr Arg Ile Met           125                 1 - #30                 1 - #35                 1 -      #40                                                                              - - CAG GAA TTT GAA AGT GAC ACG TTT TTC CCA GA - #A ATT GAT TTG GGG        AAA      480                                                                    Gln Glu Phe Glu Ser Asp Thr Phe Phe Pro Gl - #u Ile Asp Leu Gly Lys                          145  - #               150  - #               155              - - TAT AAA CTT CTC CCA GAA TAC CCA GGC GTC CT - #C TCT GAG GTC CAG GAG          528                                                                       Tyr Lys Leu Leu Pro Glu Tyr Pro Gly Val Le - #u Ser Glu Val Gln Glu                       160      - #           165      - #           170                  - - GAA AAA GGC ATC AAG TAT AAG TTT GAA GTC TA - #C GAG AAG AAA GAC              57 - #3                                                                   Glu Lys Gly Ile Lys Tyr Lys Phe Glu Val Ty - #r Glu Lys Lys Asp                       175          - #       180          - #       185                      - - TAACAGGAAG ATGCTTTCAA GTTCTCTGCT CCCCTCCTAA AGCTATGCAT TT -             #TTATAAGA    633                                                                 - - CCATGGGACT TTTGCTGGCT TTAGATCTAT GCGGCCGC      - #                      - #    671                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 187 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - Met Val Arg Pro Leu Asn Cys Ile Val Ala Va - #l Ser Gln Asn Met Gly        1               5 - #                 10 - #                 15              - - Ile Gly Lys Asn Gly Asp Leu Pro Trp Pro Pr - #o Leu Arg Asn Glu Phe                   20     - #             25     - #             30                  - - Lys Tyr Phe Gln Arg Met Thr Thr Thr Ser Se - #r Val Glu Gly Lys Gln               35         - #         40         - #         45                      - - Asn Leu Val Ile Met Gly Arg Lys Thr Trp Ph - #e Ser Ile Pro Glu Lys           50             - #     55             - #     60                          - - Asn Arg Pro Leu Lys Asp Arg Ile Asn Ile Va - #l Leu Ser Arg Glu Leu       65                 - # 70                 - # 75                 - # 80       - - Lys Glu Pro Pro Arg Gly Ala His Phe Leu Al - #a Lys Ser Leu Asp Asp                       85 - #                 90 - #                 95              - - Ala Leu Arg Leu Ile Glu Gln Pro Glu Leu Al - #a Ser Lys Val Asp Met                  100      - #           105      - #           110                  - - Val Trp Ile Val Gly Gly Ser Ser Val Tyr Gl - #n Glu Ala Met Asn Gln              115          - #       120          - #       125                      - - Pro Gly His Leu Arg Leu Phe Val Thr Arg Il - #e Met Gln Glu Phe Glu          130              - #   135              - #   140                          - - Ser Asp Thr Phe Phe Pro Glu Ile Asp Leu Gl - #y Lys Tyr Lys Leu Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Pro Glu Tyr Pro Gly Val Leu Ser Glu Val Gl - #n Glu Glu Lys Gly        Ile                                                                                             165  - #               170  - #               175             - - Lys Tyr Lys Phe Glu Val Tyr Glu Lys Lys As - #p                                      180      - #           185                                         - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - ATATATCTAG ACCACCATGC CTGGCTCAGC ACTG       - #                  -      #        34                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - ATTATTGCGG CCGCTTAGCT TTTCATTTTG ATCAT       - #                       - #       35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 134 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - GGTCTAGAGC CAAATAAAGG AAGTGGAACC ACTTCAGGTA CTACCCGTCT TC -            #TATCTGGG     60                                                                 - - CACACGTGTT TCACGTTGAC AGGTTTGCTT GGGACGCTAG TAACCATGGG CT -            #TGCTGACT    120                                                                 - - TAGGCATCGA ATTC              - #                  - #                      - #    134                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 134 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - GAATTCGATG CCTAAGTCAG CAAGCCCATG GTTACTAGCG TCCCAAGCAA AC -             #CTGTCAAC     60                                                                 - - GTGAAACACG TGTGCCCAGA TAGAAGACGG GTAGTACCTG AAGTGGTTCC AC -            #TTCCTTTA    120                                                                 - - TTTGGCTCTA GACC              - #                  - #                      - #    134                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 300 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - TAATACGACT CACTATAGGG CGAATTGGAG CTCCACCGCG GTGGCGGCCG CT -             #CTAGAACT     60                                                                 - - AGTGGATCCC CCGGGCTGCA GGAATTCGAT GGTCTAGAGC CAAATAAAGG AA -            #GTGGAACC    120                                                                 - - ACTTCAGGTA CTACCCGTCT TCTATCTGGG CACACGTGTT TCACGTTGAC AG -            #GTTTGCTT    180                                                                 - - GGGACGCTAG TAACCATGGG CTTGCTGACT TAGGCATCGA ATTCATCAAG CT -            #TATCGATA    240                                                                 - - CCGTCGACCT CGAGGGGGGG CCCGGTACCC AGCTTTTGTT CCCTTTAGTG AG -            #GGTTAATT    300                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - - CCACTTCCTT TATTTGGGAG AGGGCTTG         - #                  - #                 28                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 747 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..744                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - - ATG GCC ATA AGT GGA GTC CCT GTG CTA GGA TT - #T TTC ATC ATA GCT GTG           48                                                                       Met Ala Ile Ser Gly Val Pro Val Leu Gly Ph - #e Phe Ile Ile Ala Val             1               5 - #                 10 - #                 15              - - CTG ATG AGC GCT CAG GAA TCA TGG GCT ATC AA - #A GAA GAA CAT GTG ATC           96                                                                       Leu Met Ser Ala Gln Glu Ser Trp Ala Ile Ly - #s Glu Glu His Val Ile                        20     - #             25     - #             30                  - - ATC CAG GCC GAG TTC TAT CTG AAT CCT GAC CA - #A TCA GGC GAG TTT ATG          144                                                                       Ile Gln Ala Glu Phe Tyr Leu Asn Pro Asp Gl - #n Ser Gly Glu Phe Met                    35         - #         40         - #         45                      - - TTT GAC TTT GAT GGT GAT GAG ATT TTC CAT GT - #G GAT ATG GCA AAG AAG          192                                                                       Phe Asp Phe Asp Gly Asp Glu Ile Phe His Va - #l Asp Met Ala Lys Lys                50             - #     55             - #     60                          - - GAG ACG GTC TGG CGG CTT GAA GAA TTT GGA CG - #A TTT GCC AGC TTT GAG          240                                                                       Glu Thr Val Trp Arg Leu Glu Glu Phe Gly Ar - #g Phe Ala Ser Phe Glu            65                 - # 70                 - # 75                 - # 80       - - GCT CAA GGT GCA TTG GCC AAC ATA GCT GTG GA - #C AAA GCC AAC TTG GAA          288                                                                       Ala Gln Gly Ala Leu Ala Asn Ile Ala Val As - #p Lys Ala Asn Leu Glu                            85 - #                 90 - #                 95              - - ATC ATG ACA AAG CGC TCC AAC TAT ACT CCG AT - #C ACC AAT GTA CCT CCA          336                                                                       Ile Met Thr Lys Arg Ser Asn Tyr Thr Pro Il - #e Thr Asn Val Pro Pro                       100      - #           105      - #           110                  - - GAG GTA ACT GTG CTC ACG AAC AGC CCT GTG GA - #A CTG AGA GAG CCC AAC          384                                                                       Glu Val Thr Val Leu Thr Asn Ser Pro Val Gl - #u Leu Arg Glu Pro Asn                   115          - #       120          - #       125                      - - GTC CTC ATC TGT TTC ATA GAC AAG TTC ACC CC - #A CCA GTG GTC AAT GTC          432                                                                       Val Leu Ile Cys Phe Ile Asp Lys Phe Thr Pr - #o Pro Val Val Asn Val               130              - #   135              - #   140                          - - ACG TGG CTT CGA AAT GGA AAA CCT GTC ACC AC - #A GGA GTG TCA GAG ACA          480                                                                       Thr Trp Leu Arg Asn Gly Lys Pro Val Thr Th - #r Gly Val Ser Glu Thr           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - GTC TTC CTG CCC AGG GAA GAC CAC CTT TTC CG - #C AAG TTC CAC TAT        CTC      528                                                                    Val Phe Leu Pro Arg Glu Asp His Leu Phe Ar - #g Lys Phe His Tyr Leu                          165  - #               170  - #               175              - - CCC TTC CTG CCC TCA ACT GAG GAC GTT TAC GA - #C TGC AGG GTG GAG CAC          576                                                                       Pro Phe Leu Pro Ser Thr Glu Asp Val Tyr As - #p Cys Arg Val Glu His                       180      - #           185      - #           190                  - - TGG GGC TTG GAT GAG CCT CTT CTC AAG CAC TG - #G GAG TTT GAT GCT CCA          624                                                                       Trp Gly Leu Asp Glu Pro Leu Leu Lys His Tr - #p Glu Phe Asp Ala Pro                   195          - #       200          - #       205                      - - AGC CCT CTC CCA AAT AAA GGA AGT GGA ACC AC - #T TCA GGT ACT ACC CGT          672                                                                       Ser Pro Leu Pro Asn Lys Gly Ser Gly Thr Th - #r Ser Gly Thr Thr Arg               210              - #   215              - #   220                          - - CTT CTA TCT GGG CAC ACG TGT TTC ACG TTG AC - #A GGT TTG CTT GGG ACG          720                                                                       Leu Leu Ser Gly His Thr Cys Phe Thr Leu Th - #r Gly Leu Leu Gly Thr           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - CTA GTA ACC ATG GGC TTG CTG ACT TAG    - #                  - #                747                                                                    Leu Val Thr Met Gly Leu Leu Thr                                                               245                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 248 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                              - - Met Ala Ile Ser Gly Val Pro Val Leu Gly Ph - #e Phe Ile Ile Ala Val        1               5 - #                 10 - #                 15              - - Leu Met Ser Ala Gln Glu Ser Trp Ala Ile Ly - #s Glu Glu His Val Ile                   20     - #             25     - #             30                  - - Ile Gln Ala Glu Phe Tyr Leu Asn Pro Asp Gl - #n Ser Gly Glu Phe Met               35         - #         40         - #         45                      - - Phe Asp Phe Asp Gly Asp Glu Ile Phe His Va - #l Asp Met Ala Lys Lys           50             - #     55             - #     60                          - - Glu Thr Val Trp Arg Leu Glu Glu Phe Gly Ar - #g Phe Ala Ser Phe Glu       65                 - # 70                 - # 75                 - # 80       - - Ala Gln Gly Ala Leu Ala Asn Ile Ala Val As - #p Lys Ala Asn Leu Glu                       85 - #                 90 - #                 95              - - Ile Met Thr Lys Arg Ser Asn Tyr Thr Pro Il - #e Thr Asn Val Pro Pro                  100      - #           105      - #           110                  - - Glu Val Thr Val Leu Thr Asn Ser Pro Val Gl - #u Leu Arg Glu Pro Asn              115          - #       120          - #       125                      - - Val Leu Ile Cys Phe Ile Asp Lys Phe Thr Pr - #o Pro Val Val Asn Val          130              - #   135              - #   140                          - - Thr Trp Leu Arg Asn Gly Lys Pro Val Thr Th - #r Gly Val Ser Glu Thr      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Val Phe Leu Pro Arg Glu Asp His Leu Phe Ar - #g Lys Phe His Tyr        Leu                                                                                             165  - #               170  - #               175             - - Pro Phe Leu Pro Ser Thr Glu Asp Val Tyr As - #p Cys Arg Val Glu His                  180      - #           185      - #           190                  - - Trp Gly Leu Asp Glu Pro Leu Leu Lys His Tr - #p Glu Phe Asp Ala Pro              195          - #       200          - #       205                      - - Ser Pro Leu Pro Asn Lys Gly Ser Gly Thr Th - #r Ser Gly Thr Thr Arg          210              - #   215              - #   220                          - - Leu Leu Ser Gly His Thr Cys Phe Thr Leu Th - #r Gly Leu Leu Gly Thr      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Leu Val Thr Met Gly Leu Leu Thr                                                          245                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                              - - CCACTTCCTT TATTTGGTGC AGATTCAG         - #                  - #                 28                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 786 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..783                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                              - - ATG GTG TGT CTG AAG CTC CCT GGA GGC TCC TG - #C ATG ACA GCG CTG ACA           48                                                                       Met Val Cys Leu Lys Leu Pro Gly Gly Ser Cy - #s Met Thr Ala Leu Thr             1               5 - #                 10 - #                 15              - - GTG ACA CTG ATG GTG CTG AGC TCC CGA CTG GC - #T TTG GCT GGG GAC ACC           96                                                                       Val Thr Leu Met Val Leu Ser Ser Arg Leu Al - #a Leu Ala Gly Asp Thr                        20     - #             25     - #             30                  - - CGA CCA CGT TTC TTG TGG CAG CTT AAG TTT GA - #A TGT CAT TTC TTC AAT          144                                                                       Arg Pro Arg Phe Leu Trp Gln Leu Lys Phe Gl - #u Cys His Phe Phe Asn                    35         - #         40         - #         45                      - - GGG ACG GAG CGG GTG CGG TTG CTG GAA AGA TG - #C ATC TAT AAC CAA GAG          192                                                                       Gly Thr Glu Arg Val Arg Leu Leu Glu Arg Cy - #s Ile Tyr Asn Gln Glu                50             - #     55             - #     60                          - - GAG TCC GTG CGC TTC GAC AGC GAC GTG GGG GA - #G TAC CGG GCG GTT GAG          240                                                                       Glu Ser Val Arg Phe Asp Ser Asp Val Gly Gl - #u Tyr Arg Ala Val Glu            65                 - # 70                 - # 75                 - # 80       - - GAG CTG GGG CGG CCT GAT GCC GAG TAC TGG AA - #C AGC CAG AAG GAC CTC          288                                                                       Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp As - #n Ser Gln Lys Asp Leu                            85 - #                 90 - #                 95              - - CTG GAG CAG AAG CGG GGC CAG GTG GAC AAT TA - #C TGC AGA CAC AAC TAC          336                                                                       Leu Glu Gln Lys Arg Gly Gln Val Asp Asn Ty - #r Cys Arg His Asn Tyr                       100      - #           105      - #           110                  - - GGG GTT GGT GAG AGC TTC ACA GTG CAG CGG CG - #A GTT GAG CCT AAG GTG          384                                                                       Gly Val Gly Glu Ser Phe Thr Val Gln Arg Ar - #g Val Glu Pro Lys Val                   115          - #       120          - #       125                      - - ACT GTG TAT CCT TCA AAG ACC CAG CCC CTG CA - #G CAC CAC AAC CTC CTG          432                                                                       Thr Val Tyr Pro Ser Lys Thr Gln Pro Leu Gl - #n His His Asn Leu Leu               130              - #   135              - #   140                          - - GTC TGC TCT GTG AGT GGT TTC TAT CCA GGC AG - #C ATT GAA GTC AGG TGG          480                                                                       Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Se - #r Ile Glu Val Arg Trp           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - TTC CGG AAC GGC CAG GAA GAG AAG GCT GGG GT - #G GTG TCC ACG GGC        CTG      528                                                                    Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Va - #l Val Ser Thr Gly Leu                          165  - #               170  - #               175              - - ATC CAG AAT GGA GAT TGG ACC TTC CAG ACC CT - #G GTG ATG CTG GAA ATA          576                                                                       Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Le - #u Val Met Leu Glu Ile                       180      - #           185      - #           190                  - - GTT CCT CGG AGT GGA GAG GTT TAC ACC TGC CA - #A GTG GAG CAC CCA AGT          624                                                                       Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gl - #n Val Glu His Pro Ser                   195          - #       200          - #       205                      - - GTG ACG AGC CCT CTC ACA GTG GAA TGG AGA GC - #A CGG TCT GAA TCT GCA          672                                                                       Val Thr Ser Pro Leu Thr Val Glu Trp Arg Al - #a Arg Ser Glu Ser Ala               210              - #   215              - #   220                          - - CCA AAT AAA GGA AGT GGA ACC ACT TCA GGT AC - #T ACC CGT CTT CTA TCT          720                                                                       Pro Asn Lys Gly Ser Gly Thr Thr Ser Gly Th - #r Thr Arg Leu Leu Ser           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - GGG CAC ACG TGT TTC ACG TTG ACA GGT TTG CT - #T GGG ACG CTA GTA        ACC      768                                                                    Gly His Thr Cys Phe Thr Leu Thr Gly Leu Le - #u Gly Thr Leu Val Thr                          245  - #               250  - #               255              - - ATG GGC TTG CTG ACT TAG         - #                  - #                      - # 786                                                                  Met Gly Leu Leu Thr                                                                       260                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 261 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                              - - Met Val Cys Leu Lys Leu Pro Gly Gly Ser Cy - #s Met Thr Ala Leu Thr        1               5 - #                 10 - #                 15              - - Val Thr Leu Met Val Leu Ser Ser Arg Leu Al - #a Leu Ala Gly Asp Thr                   20     - #             25     - #             30                  - - Arg Pro Arg Phe Leu Trp Gln Leu Lys Phe Gl - #u Cys His Phe Phe Asn               35         - #         40         - #         45                      - - Gly Thr Glu Arg Val Arg Leu Leu Glu Arg Cy - #s Ile Tyr Asn Gln Glu           50             - #     55             - #     60                          - - Glu Ser Val Arg Phe Asp Ser Asp Val Gly Gl - #u Tyr Arg Ala Val Glu       65                 - # 70                 - # 75                 - # 80       - - Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp As - #n Ser Gln Lys Asp Leu                       85 - #                 90 - #                 95              - - Leu Glu Gln Lys Arg Gly Gln Val Asp Asn Ty - #r Cys Arg His Asn Tyr                  100      - #           105      - #           110                  - - Gly Val Gly Glu Ser Phe Thr Val Gln Arg Ar - #g Val Glu Pro Lys Val              115          - #       120          - #       125                      - - Thr Val Tyr Pro Ser Lys Thr Gln Pro Leu Gl - #n His His Asn Leu Leu          130              - #   135              - #   140                          - - Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Se - #r Ile Glu Val Arg Trp      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Va - #l Val Ser Thr Gly        Leu                                                                                             165  - #               170  - #               175             - - Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Le - #u Val Met Leu Glu Ile                  180      - #           185      - #           190                  - - Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gl - #n Val Glu His Pro Ser              195          - #       200          - #       205                      - - Val Thr Ser Pro Leu Thr Val Glu Trp Arg Al - #a Arg Ser Glu Ser Ala          210              - #   215              - #   220                          - - Pro Asn Lys Gly Ser Gly Thr Thr Ser Gly Th - #r Thr Arg Leu Leu Ser      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Gly His Thr Cys Phe Thr Leu Thr Gly Leu Le - #u Gly Thr Leu Val        Thr                                                                                             245  - #               250  - #               255             - - Met Gly Leu Leu Thr                                                                  260                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 189 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..186                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                              - - TTG GAT CCA CGA TCG TTT CTA TTG CGC AAT CC - #A AAT GAT AAG TAC GAA           48                                                                       Leu Asp Pro Arg Ser Phe Leu Leu Arg Asn Pr - #o Asn Asp Lys Tyr Glu             1               5 - #                 10 - #                 15              - - CCA TTT TGG GAA GAT ACT ACA GAG AAC GTG GT - #G TGT GCC CTG GGC CTG           96                                                                       Pro Phe Trp Glu Asp Thr Thr Glu Asn Val Va - #l Cys Ala Leu Gly Leu                        20     - #             25     - #             30                  - - ACT GTG GGT CTG GTG GGC ATC ATT ATT GGG AC - #C ATC TTC ATC ATC AAG          144                                                                       Thr Val Gly Leu Val Gly Ile Ile Ile Gly Th - #r Ile Phe Ile Ile Lys                    35         - #         40         - #         45                      - - GGA GTG CGC AAA AGC AAT GCA GCA GAA CGC AG - #G GGG CCT CTG                 - # 186                                                                    Gly Val Arg Lys Ser Asn Ala Ala Glu Arg Ar - #g Gly Pro Leu                        50             - #     55             - #     60                          - - TAA                  - #                  - #                  - #                189                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 62 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                              - - Leu Asp Pro Arg Ser Phe Leu Leu Arg Asn Pr - #o Asn Asp Lys Tyr Glu        1               5 - #                 10 - #                 15              - - Pro Phe Trp Glu Asp Thr Thr Glu Asn Val Va - #l Cys Ala Leu Gly Leu                   20     - #             25     - #             30                  - - Thr Val Gly Leu Val Gly Ile Ile Ile Gly Th - #r Ile Phe Ile Ile Lys               35         - #         40         - #         45                      - - Gly Val Arg Lys Ser Asn Ala Ala Glu Arg Ar - #g Gly Pro Leu                   50             - #     55             - #     60                          - -  - - (2) INFORMATION FOR SEQ ID NO:33:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 192 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..189                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                              - - TTG GAT CCA CGA TCG TTT CTA TTG CGC AAT CC - #A AAT GAT AAG TAC GAA           48                                                                       Leu Asp Pro Arg Ser Phe Leu Leu Arg Asn Pr - #o Asn Asp Lys Tyr Glu             1               5 - #                 10 - #                 15              - - CCA TTT TGG GAA GAT CAG AGC AAG ATG CTG AG - #T GGA GTC GGG GGC TTC           96                                                                       Pro Phe Trp Glu Asp Gln Ser Lys Met Leu Se - #r Gly Val Gly Gly Phe                        20     - #             25     - #             30                  - - GTG CTG GGC CTG CTC TTC CTT GGG GCC GGG CT - #G TTC ATC TAC TTC AGG          144                                                                       Val Leu Gly Leu Leu Phe Leu Gly Ala Gly Le - #u Phe Ile Tyr Phe Arg                    35         - #         40         - #         45                      - - AAT CAG AAA GGA CAC TCT GGA CTT CAG CCA AC - #A GGA TTC CTG AGC              18 - #9                                                                   Asn Gln Lys Gly His Ser Gly Leu Gln Pro Th - #r Gly Phe Leu Ser                    50             - #     55             - #     60                          - - TGA                  - #                  - #                  - #                192                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 63 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                              - - Leu Asp Pro Arg Ser Phe Leu Leu Arg Asn Pr - #o Asn Asp Lys Tyr Glu        1               5 - #                 10 - #                 15              - - Pro Phe Trp Glu Asp Gln Ser Lys Met Leu Se - #r Gly Val Gly Gly Phe                   20     - #             25     - #             30                  - - Val Leu Gly Leu Leu Phe Leu Gly Ala Gly Le - #u Phe Ile Tyr Phe Arg               35         - #         40         - #         45                      - - Asn Gln Lys Gly His Ser Gly Leu Gln Pro Th - #r Gly Phe Leu Ser               50             - #     55             - #     60                          - -  - - (2) INFORMATION FOR SEQ ID NO:35:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                              - - CGATCGTGGA TCCAAGTTTA GGTTCGTATC TGTTTCAAA      - #                      - #    39                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:36:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                              - - CGATCGAGGA TCCAAGATGG TGGCAGACAG GACC       - #                  -      #        34                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:37:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                              - - ACGCGTCCAC CATGGCCATA AGTGGAGTCC CT       - #                  - #              32                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:38:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                              - - GGATCCAACT CTGTAGTCTC TGGGAGAG         - #                  - #                 28                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:39:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                              - - ACGCGTCCAC CATGGTGTGT CTGAAGCTCC TG       - #                  - #              32                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:40:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                              - - GGATCCAACT TGCTCTGTGC AGATTCAGA         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:41:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 292 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                              - - GAATTCTTTT TTGCGTGTGG CAGTTTTAAG TTATTAGTTT TTAAAATCAG TA -             #CTTTTTAA     60                                                                 - - TGGAAACAAC TTGACCAAAA ATTTGTCACA GAATTTTGAG ACCCATTAAA AA -            #AGTTAAAT    120                                                                 - - GAGAAACCTG TGTGTTCCTT TGGTCAACAC CGAGACATTT AGGTGAAAGA CA -            #TCTAATTC    180                                                                 - - TGGTTTTACG AATCTGGAAA CTTCTTGAAA ATGTAATTCT TGAGTTAACA CT -            #TCTGGGTG    240                                                                 - - GAGAATAGGG TTGTTTTCCC CCCACATAAT TGGAAGGGGA AGGAATATCG AT - #                292                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:42:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                              - - TCGATGGCGC GCCTTAATTA            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:43:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                              - - AGCTTAATTA AGGCGCGCCA            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:44:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1147 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 7..1137                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                              - - GCGGCC GCG TCG ACC AAG GGC CCC AGC GTG TTC - #CCC CTG GCC CCC TGC            48                                                                               Ala Ser Thr Lys Gly Pro Se - #r Val Phe Pro Leu Ala Pro Cys                     1         - #      5            - #      10                           - - TCC CGC AGC ACC AGC GGC GGC ACC GCC GCC CT - #G GGC TGC CTG GTG AAG           96                                                                       Ser Arg Ser Thr Ser Gly Gly Thr Ala Ala Le - #u Gly Cys Leu Val Lys            15                 - # 20                 - # 25                 - # 30       - - GAC TAC TTC CCC GAG CCC GTG ACC GTG AGC TG - #G AAC AGC GGC GCC CTG          144                                                                       Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Tr - #p Asn Ser Gly Ala Leu                            35 - #                 40 - #                 45              - - ACC AGC GGC GTC CAC ACC TTC CCC GCC GTG CT - #G CAG TCC AGC GGC CTG          192                                                                       Thr Ser Gly Val His Thr Phe Pro Ala Val Le - #u Gln Ser Ser Gly Leu                        50     - #             55     - #             60                  - - TAC TCC CTG AGC AGC GTG GTG ACC GTG CCC AG - #C AGC AGC CTG GGC ACC          240                                                                       Tyr Ser Leu Ser Ser Val Val Thr Val Pro Se - #r Ser Ser Leu Gly Thr                    65         - #         70         - #         75                      - - CAG ACC TAC ACC TGC AAC GTG AAC CAC AAG CC - #C AGC AAC ACC AAG GTG          288                                                                       Gln Thr Tyr Thr Cys Asn Val Asn His Lys Pr - #o Ser Asn Thr Lys Val                80             - #     85             - #     90                          - - GAC AAG CGC GTG GAG CTG AAG ACC CCC CTG GG - #C GAC ACC ACC CAC ACC          336                                                                       Asp Lys Arg Val Glu Leu Lys Thr Pro Leu Gl - #y Asp Thr Thr His Thr            95                 - #100                 - #105                 - #110       - - TGC CCC CGC TGC CCC GAG CCC AAG AGC TGC GA - #C ACC CCT CCC CCC TGC          384                                                                       Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys As - #p Thr Pro Pro Pro Cys                           115  - #               120  - #               125              - - CCC CGC TGC CCC GAG CCC AAG AGC TGC GAC AC - #C CCT CCC CCC TGC CCC          432                                                                       Pro Arg Cys Pro Glu Pro Lys Ser Cys Asp Th - #r Pro Pro Pro Cys Pro                       130      - #           135      - #           140                  - - CGC TGC CCC GAG CCC AAG AGC TGC GAC ACC CC - #T CCC CCC TGC CCC CGC          480                                                                       Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pr - #o Pro Pro Cys Pro Arg                   145          - #       150          - #       155                      - - TGC CCC GCC CCC GAG CTG CTG GGC GGC CCC AG - #C GTG TTC CTG TTC CCC          528                                                                       Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Se - #r Val Phe Leu Phe Pro               160              - #   165              - #   170                          - - CCC AAG CCC AAG GAC ACC CTG ATG ATC TCC CG - #C ACC CCC GAG GTG ACC          576                                                                       Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Ar - #g Thr Pro Glu Val Thr           175                 1 - #80                 1 - #85                 1 -      #90                                                                              - - TGC GTG GTG GTG GAC GTG AGC CAC GAG GAC CC - #C GAG GTG CAG TTC        AAG      624                                                                    Cys Val Val Val Asp Val Ser His Glu Asp Pr - #o Glu Val Gln Phe Lys                          195  - #               200  - #               205              - - TGG TAC GTG GAC GGC GTG GAG GTG CAT AAC GC - #C AAG ACC AAG CCC CGC          672                                                                       Trp Tyr Val Asp Gly Val Glu Val His Asn Al - #a Lys Thr Lys Pro Arg                       210      - #           215      - #           220                  - - GAG GAG CAG TAC AAC AGC ACC TTC CGC GTG GT - #G AGC GTG CTG ACC GTG          720                                                                       Glu Glu Gln Tyr Asn Ser Thr Phe Arg Val Va - #l Ser Val Leu Thr Val                   225          - #       230          - #       235                      - - CTG CAC CAG GAC TGG CTG AAC GGC AAG GAG TA - #C AAG TGC AAG GTG AGC          768                                                                       Leu His Gln Asp Trp Leu Asn Gly Lys Glu Ty - #r Lys Cys Lys Val Ser               240              - #   245              - #   250                          - - AAC AAG GCC CTG CCC GCC CCC ATC GAG AAG AC - #C ATC TCC AAG ACC AAG          816                                                                       Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Th - #r Ile Ser Lys Thr Lys           255                 2 - #60                 2 - #65                 2 -      #70                                                                              - - GGC CAG CCC CGC GAG CCC CAG GTG TAC ACC CT - #G CCC CCC AGC CGC        GAG      864                                                                    Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Le - #u Pro Pro Ser Arg Glu                          275  - #               280  - #               285              - - GAG ATG ACC AAG AAC CAG GTG AGC CTG ACC TG - #C CTG GTG AAG GGC TTC          912                                                                       Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cy - #s Leu Val Lys Gly Phe                       290      - #           295      - #           300                  - - TAC CCC AGC GAC ATC GCC GTG GAG TGG GAG AG - #C AGC GGC CAG CCC GAG          960                                                                       Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Se - #r Ser Gly Gln Pro Glu                   305          - #       310          - #       315                      - - AAC AAC TAC AAC ACC ACC CCC CCC ATG CTG GA - #C AGC GAC GGC AGC TTC         1008                                                                       Asn Asn Tyr Asn Thr Thr Pro Pro Met Leu As - #p Ser Asp Gly Ser Phe               320              - #   325              - #   330                          - - TTC CTG TAC AGC AAG CTG ACC GTG GAC AAG AG - #C CGC TGG CAG CAG GGC         1056                                                                       Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Se - #r Arg Trp Gln Gln Gly           335                 3 - #40                 3 - #45                 3 -      #50                                                                              - - AAC ATC TTC TCC TGC AGC GTG ATG CAT GAG GC - #C CTG CAC AAC CGC        TTC     1104                                                                    Asn Ile Phe Ser Cys Ser Val Met His Glu Al - #a Leu His Asn Arg Phe                          355  - #               360  - #               365              - - ACC CAG AAG AGC CTG AGC CTG AGC CCC GGC AA - #G TGATAGATCT                  - #1147                                                                    Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ly - #s                                           370      - #           375                                         - -  - - (2) INFORMATION FOR SEQ ID NO:45:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 377 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                              - - Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Le - #u Ala Pro Cys Ser Arg        1               5 - #                 10 - #                 15              - - Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cy - #s Leu Val Lys Asp Tyr                   20     - #             25     - #             30                  - - Phe Pro Glu Pro Val Thr Val Ser Trp Asn Se - #r Gly Ala Leu Thr Ser               35         - #         40         - #         45                      - - Gly Val His Thr Phe Pro Ala Val Leu Gln Se - #r Ser Gly Leu Tyr Ser           50             - #     55             - #     60                          - - Leu Ser Ser Val Val Thr Val Pro Ser Ser Se - #r Leu Gly Thr Gln Thr       65                 - # 70                 - # 75                 - # 80       - - Tyr Thr Cys Asn Val Asn His Lys Pro Ser As - #n Thr Lys Val Asp Lys                       85 - #                 90 - #                 95              - - Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Th - #r Thr His Thr Cys Pro                  100      - #           105      - #           110                  - - Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pr - #o Pro Pro Cys Pro Arg              115          - #       120          - #       125                      - - Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pr - #o Pro Cys Pro Arg Cys          130              - #   135              - #   140                          - - Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pr - #o Cys Pro Arg Cys Pro      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Ph - #e Leu Phe Pro Pro        Lys                                                                                             165  - #               170  - #               175             - - Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pr - #o Glu Val Thr Cys Val                  180      - #           185      - #           190                  - - Val Val Asp Val Ser His Glu Asp Pro Glu Va - #l Gln Phe Lys Trp Tyr              195          - #       200          - #       205                      - - Val Asp Gly Val Glu Val His Asn Ala Lys Th - #r Lys Pro Arg Glu Glu          210              - #   215              - #   220                          - - Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Va - #l Leu Thr Val Leu His      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cy - #s Lys Val Ser Asn        Lys                                                                                             245  - #               250  - #               255             - - Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Se - #r Lys Thr Lys Gly Gln                  260      - #           265      - #           270                  - - Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pr - #o Ser Arg Glu Glu Met              275          - #       280          - #       285                      - - Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Va - #l Lys Gly Phe Tyr Pro          290              - #   295              - #   300                          - - Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gl - #y Gln Pro Glu Asn Asn      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser As - #p Gly Ser Phe Phe        Leu                                                                                             325  - #               330  - #               335             - - Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Tr - #p Gln Gln Gly Asn Ile                  340      - #           345      - #           350                  - - Phe Ser Cys Ser Val Met His Glu Ala Leu Hi - #s Asn Arg Phe Thr Gln              355          - #       360          - #       365                      - - Lys Ser Leu Ser Leu Ser Pro Gly Lys                                          370              - #   375                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:46:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 999 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 9..989                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                              - - GCGGCCGC GCG TCG ACC AAG GGC CCC AGC GTG TTC - #CCC CTG GCC CCC TGC          50                                                                                 Ala Ser Thr Lys Gly P - #ro Ser Val Phe Pro Leu Ala Pro Cys                     1       - #        5          - #        10                         - - AGC CGC AGC ACC AGC GAG AGC ACC GCC GCC CT - #G GGC TGC CTG GTG AAG           98                                                                       Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Le - #u Gly Cys Leu Val Lys            15                 - # 20                 - # 25                 - # 30       - - GAC TAC TTC CCC GAG CCC GTG ACC GTG AGC TG - #G AAC AGC GGC GCC CTG          146                                                                       Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Tr - #p Asn Ser Gly Ala Leu                            35 - #                 40 - #                 45              - - ACC AGC GGC GTG CAC ACC TTC CCC GCC GTG CT - #G CAG AGC AGC GGC CTG          194                                                                       Thr Ser Gly Val His Thr Phe Pro Ala Val Le - #u Gln Ser Ser Gly Leu                        50     - #             55     - #             60                  - - TAC TCC CTG AGC AGC GTG GTG ACC GTG CCC AG - #C AGC AGC CTG GGC ACC          242                                                                       Tyr Ser Leu Ser Ser Val Val Thr Val Pro Se - #r Ser Ser Leu Gly Thr                    65         - #         70         - #         75                      - - AAG ACC TAC ACC TGC AAC GTG GAC CAC AAG CC - #C AGC AAC ACC AAG GTG          290                                                                       Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pr - #o Ser Asn Thr Lys Val                80             - #     85             - #     90                          - - GAC AAG CGC GTG GAG AGC AAG TAC GGC CCC CC - #C TGC CCC AGC TGC CCC          338                                                                       Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pr - #o Cys Pro Ser Cys Pro            95                 - #100                 - #105                 - #110       - - GCC CCC GAG TTC CTG GGC GGC CCC AGC GTG TT - #C CTG TTC CCC CCC AAG          386                                                                       Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Ph - #e Leu Phe Pro Pro Lys                           115  - #               120  - #               125              - - CCC AAG GAC ACC CTG ATG ATC AGC CGC ACC CC - #C GAG GTG ACC TGC GTG          434                                                                       Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pr - #o Glu Val Thr Cys Val                       130      - #           135      - #           140                  - - GTG GTG GAC GTG AGC CAG GAG GAC CCC GAG GT - #G CAG TTC AAC TGG TAC          482                                                                       Val Val Asp Val Ser Gln Glu Asp Pro Glu Va - #l Gln Phe Asn Trp Tyr                   145          - #       150          - #       155                      - - GTG GAC GGC GTG GAG GTG CAT AAC GCC AAG AC - #C AAG CCC CGC GAG GAG          530                                                                       Val Asp Gly Val Glu Val His Asn Ala Lys Th - #r Lys Pro Arg Glu Glu               160              - #   165              - #   170                          - - CAG TTC AAC AGC ACC TAC CGC GTG GTG AGC GT - #G CTG ACC GTG CTG CAC          578                                                                       Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Va - #l Leu Thr Val Leu His           175                 1 - #80                 1 - #85                 1 -      #90                                                                              - - CAG GAC TGG CTG AAC GGC AAG GAG TAC AAG TG - #C AAG GTG TCC AAC        AAG      626                                                                    Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cy - #s Lys Val Ser Asn Lys                          195  - #               200  - #               205              - - GGC CTG CCC AGC AGC ATC GAG AAG ACC ATC AG - #C AAG GCC AAG GGC CAG          674                                                                       Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Se - #r Lys Ala Lys Gly Gln                       210      - #           215      - #           220                  - - CCC CGC GAG CCC CAG GTG TAC ACC CTG CCC CC - #C AGC CAG GAG GAG ATG          722                                                                       Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pr - #o Ser Gln Glu Glu Met                   225          - #       230          - #       235                      - - ACC AAG AAC CAG GTG AGC CTG ACC TGC CTG GT - #G AAG GGC TTC TAC CCC          770                                                                       Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Va - #l Lys Gly Phe Tyr Pro               240              - #   245              - #   250                          - - AGC GAC ATC GCC GTG GAG TGG GAG AGC AAC GG - #C CAG CCC GAG AAC AAC          818                                                                       Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gl - #y Gln Pro Glu Asn Asn           255                 2 - #60                 2 - #65                 2 -      #70                                                                              - - TAC AAG ACC ACC CCC CCC GTG CTG GAC AGC GA - #C GGC AGC TTC TTC        CTG      866                                                                    Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser As - #p Gly Ser Phe Phe Leu                          275  - #               280  - #               285              - - TAC AGC CGC CTG ACC GTG GAC AAG AGC CGC TG - #G CAG GAG GGC AAC GTG          914                                                                       Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Tr - #p Gln Glu Gly Asn Val                       290      - #           295      - #           300                  - - TTC TCC TGC TCC GTG ATG CAT GAG GCC CTG CA - #C AAC CAC TAC ACC CAG          962                                                                       Phe Ser Cys Ser Val Met His Glu Ala Leu Hi - #s Asn His Tyr Thr Gln                   305          - #       310          - #       315                      - - AAG AGC CTG AGC CTG AGC CTG GGC AAG TGATAGATC - #T                      - #     999                                                                    Lys Ser Leu Ser Leu Ser Leu Gly Lys                                               320              - #   325                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:47:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 327 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                              - - Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Le - #u Ala Pro Cys Ser Arg        1               5 - #                 10 - #                 15              - - Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cy - #s Leu Val Lys Asp Tyr                   20     - #             25     - #             30                  - - Phe Pro Glu Pro Val Thr Val Ser Trp Asn Se - #r Gly Ala Leu Thr Ser               35         - #         40         - #         45                      - - Gly Val His Thr Phe Pro Ala Val Leu Gln Se - #r Ser Gly Leu Tyr Ser           50             - #     55             - #     60                          - - Leu Ser Ser Val Val Thr Val Pro Ser Ser Se - #r Leu Gly Thr Lys Thr       65                 - # 70                 - # 75                 - # 80       - - Tyr Thr Cys Asn Val Asp His Lys Pro Ser As - #n Thr Lys Val Asp Lys                       85 - #                 90 - #                 95              - - Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pr - #o Ser Cys Pro Ala Pro                  100      - #           105      - #           110                  - - Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Ph - #e Pro Pro Lys Pro Lys              115          - #       120          - #       125                      - - Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Va - #l Thr Cys Val Val Val          130              - #   135              - #   140                          - - Asp Val Ser Gln Glu Asp Pro Glu Val Gln Ph - #e Asn Trp Tyr Val Asp      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Gly Val Glu Val His Asn Ala Lys Thr Lys Pr - #o Arg Glu Glu Gln        Phe                                                                                             165  - #               170  - #               175             - - Asn Ser Thr Tyr Arg Val Val Ser Val Leu Th - #r Val Leu His Gln Asp                  180      - #           185      - #           190                  - - Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Va - #l Ser Asn Lys Gly Leu              195          - #       200          - #       205                      - - Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Al - #a Lys Gly Gln Pro Arg          210              - #   215              - #   220                          - - Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gl - #n Glu Glu Met Thr Lys      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gl - #y Phe Tyr Pro Ser        Asp                                                                                             245  - #               250  - #               255             - - Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pr - #o Glu Asn Asn Tyr Lys                  260      - #           265      - #           270                  - - Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Se - #r Phe Phe Leu Tyr Ser              275          - #       280          - #       285                      - - Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Gl - #u Gly Asn Val Phe Ser          290              - #   295              - #   300                          - - Cys Ser Val Met His Glu Ala Leu His Asn Hi - #s Tyr Thr Gln Lys Ser      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Leu Ser Leu Ser Leu Gly Lys                                                              325                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:48:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 337 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 9..326                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                              - - GCGGCCGC ACT GTG GCT GCA CCA TCT GTC TTC ATC - #TTC CCG CCA TCT        GAT      50                                                                              Thr Val Ala Ala Pro S - #er Val Phe Ile Phe Pro Pro Ser Asp                    1       - #        5          - #        10                         - - GAG CAG CTT AAG TCC GGA ACC GCC AGC GTG GT - #G TGC CTG CTG AAC AAC           98                                                                       Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Va - #l Cys Leu Leu Asn Asn            15                 - # 20                 - # 25                 - # 30       - - TTC TAC CCC CGC GAG GCC AAG GTG CAG TGG AA - #G GTG GAC AAC GCC CTC          146                                                                       Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Ly - #s Val Asp Asn Ala Leu                            35 - #                 40 - #                 45              - - CAG AGC GGC AAC TCC CAG GAG AGC GTG ACC GA - #G CAG GAC AGC AAG GAC          194                                                                       Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Gl - #u Gln Asp Ser Lys Asp                        50     - #             55     - #             60                  - - AGC ACC TAC AGC CTG AGC AGC ACC CTG ACC CT - #G AGC AAG GCC GAC TAC          242                                                                       Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Le - #u Ser Lys Ala Asp Tyr                    65         - #         70         - #         75                      - - GAG AAG CAC AAG GTG TAC GCC TGC GAG GTG AC - #C CAT CAG GGC CTG AGC          290                                                                       Glu Lys His Lys Val Tyr Ala Cys Glu Val Th - #r His Gln Gly Leu Ser                80             - #     85             - #     90                          - - AGC CCC GTG ACC AAG AGC TTC AAC CGG GGC GA - #G TGC TAGTGAGATC               336                                                                       Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Gl - #u Cys                            95                 - #100                 - #105                              - - T                  - #                  - #                  - #                  337                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:49:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 106 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                              - - Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pr - #o Pro Ser Asp Glu Gln        1               5 - #                 10 - #                 15              - - Leu Lys Ser Gly Thr Ala Ser Val Val Cys Le - #u Leu Asn Asn Phe Tyr                   20     - #             25     - #             30                  - - Pro Arg Glu Ala Lys Val Gln Trp Lys Val As - #p Asn Ala Leu Gln Ser               35         - #         40         - #         45                      - - Gly Asn Ser Gln Glu Ser Val Thr Glu Gln As - #p Ser Lys Asp Ser Thr           50             - #     55             - #     60                          - - Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Ly - #s Ala Asp Tyr Glu Lys       65                 - # 70                 - # 75                 - # 80       - - His Lys Val Tyr Ala Cys Glu Val Thr His Gl - #n Gly Leu Ser Ser Pro                       85 - #                 90 - #                 95              - - Val Thr Lys Ser Phe Asn Arg Gly Glu Cys                                              100      - #           105                                         - -  - - (2) INFORMATION FOR SEQ ID NO:50:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 346 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 9..335                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                              - - GCGGCCGC ACC GTC CTA GGT CAG CCC AAG GCG GCG - #CCC AGC GTG ACC CTG          50                                                                                 Thr Val Leu Gly Gln P - #ro Lys Ala Ala Pro Ser Val Thr Leu                     1       - #        5          - #        10                         - - TTC CCC CCC AGC AGC GAG GAG CTG CAG GCC AA - #C AAG GCC ACC CTG GTG           98                                                                       Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala As - #n Lys Ala Thr Leu Val            15                 - # 20                 - # 25                 - # 30       - - TGC CTG ATC AGC GAC TTC TAC CCC GGG GCC GT - #G ACC GTG GCC TGG AAG          146                                                                       Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Va - #l Thr Val Ala Trp Lys                            35 - #                 40 - #                 45              - - GCC GAC AGC AGC CCC GTG AAG GCC GGC GTG GA - #G ACC ACC ACC CCC AGC          194                                                                       Ala Asp Ser Ser Pro Val Lys Ala Gly Val Gl - #u Thr Thr Thr Pro Ser                        50     - #             55     - #             60                  - - AAG CAG AGC AAC AAC AAG TAC GCC GCC AGC AG - #C TAC CTG AGC CTG ACC          242                                                                       Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Se - #r Tyr Leu Ser Leu Thr                    65         - #         70         - #         75                      - - CCC GAG CAG TGG AAG AGC CAC CGC AGC TAC AG - #C TGC CAG GTC ACC CAC          290                                                                       Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Se - #r Cys Gln Val Thr His                80             - #     85             - #     90                          - - GAG GGC AGC ACC GTG GAG AAG ACC GTG GCC CC - #C ACC GAG TGC AGC              33 - #5                                                                   Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pr - #o Thr Glu Cys Ser                95                 - #100                 - #105                              - - TAGTGAGATC T               - #                  - #                      - #      346                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:51:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 109 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                              - - Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Se - #r Val Thr Leu Phe Pro        1               5 - #                 10 - #                 15              - - Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Al - #a Thr Leu Val Cys Leu                   20     - #             25     - #             30                  - - Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Va - #l Ala Trp Lys Ala Asp               35         - #         40         - #         45                      - - Ser Ser Pro Val Lys Ala Gly Val Glu Thr Th - #r Thr Pro Ser Lys Gln           50             - #     55             - #     60                          - - Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Le - #u Ser Leu Thr Pro Glu       65                 - # 70                 - # 75                 - # 80       - - Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gl - #n Val Thr His Glu Gly                       85 - #                 90 - #                 95              - - Ser Thr Val Glu Lys Thr Val Ala Pro Thr Gl - #u Cys Ser                              100      - #           105                                         - -  - - (2) INFORMATION FOR SEQ ID NO:52:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                              - - TCTAGAATTC ACGCGTCCAC CATGGACTGG ACCTGGAG      - #                      - #     38                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:53:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                              - - TCTAGAATTC ACGCGTCCAC CATGGACACA CTTTGCTACA C    - #                      - #   41                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:54:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                              - - TCTAGAATTC ACGCGTCCAC CATGGAGTTT GGGCTGAGCT GG    - #                      - #  42                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:55:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                              - - TCTAGAATTC ACGCGTCCAC CATGAAACAC CTGTGGTTCT TCCT   - #                      - # 44                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:56:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                              - - TCTAGAATTC ACGCGTCCAC CATGGGGTCA ACCGCCATCC T    - #                      - #   41                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:57:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                              - - TCTAGAATTC ACGCGTCCAC CATGTCTGTC TCCTTCCTCA TCTT   - #                      - # 44                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:58:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                              - - GCCTGAGTTC CACGACACCG TCAC          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:59:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                              - - GGGGAAAAGG GTTGGGGCGG ATGC          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:60:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                              - - GAGGGGCCCT TGGTCGACGC TGAGGAGACG GTGACCAGG      - #                      - #    39                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:61:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                              - - GAGGGGCCCT TGGTCGACGC TGAAGAGACG GTGACCATTG     - #                      - #    40                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:62:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                              - - GAGGGGCCCT TGGTCGACGC TGAGGAGACG GTGACCGTG      - #                      - #    39                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:63:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                              - - TCTAGAATTC ACGCGTCCAC CATGGACATG AGGGTCCCCG CTCAG   - #                      - #45                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:64:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                              - - TCTAGAATTC ACGCGTCCAC CATGAGGCTC CCTGCTCAGC     - #                      - #    40                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:65:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                              - - TCTAGAATTC ACGCGTCCAC CATGGAAGCC CCAGCGCAGC TT    - #                      - #  42                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:66:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                              - - TCTAGAATTC ACGCGTCCAC CATGGTGTTG CAGACCCAGG T    - #                      - #   41                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:67:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                              - - TCTAGAATTC ACGCGTCCAC CATGGGGTCC CAGGTTCACC T    - #                      - #   41                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:68:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                              - - TCTAGAATTC ACGCGTCCAC CATGTTGCCA TCACAACTCA TTG    - #                      - # 43                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:69:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                              - - TCTAGAATTC ACGCGTCCAC CATGGTGTCC CCGTTGCAAT T    - #                      - #   41                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:70:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                              - - GGTTCCGGAC TTAAGCTGCT CATCAGATGG CGGG       - #                  -      #        34                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:71:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                              - - TCTAGAATTC ACGCGTCCAC CATGGCCTGC TCTCCTCTCC TCCT   - #                      - # 44                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:72:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                              - - TCTAGAATTC ACGCGTCCAC CATGGCCTGG GCTCTGCTGC TCCT   - #                      - # 44                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:73:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                              - - TCTAGAATTC ACGCGTCCAC CATGGCCTGG ATCCTTCTCC TCCTC   - #                      - #45                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:74:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                              - - TCTAGAATTC ACGCGTCCAC CATGGCCTGG ACCCCTCTCT GGCTC   - #                      - #45                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:75:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                              - - TCTAGAATTC ACGCGTCCAC CATGGCCTGG GCCCCACTAC T    - #                      - #   41                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:76:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                              - - TCTAGAATTC ACGCGTCCAC CATGGCCTGG ATGATGCTTC TCCT   - #                      - # 44                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:77:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                              - - GGCGCCGCCT TGGGCTGACC TAGGACGGT         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:78:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "DNA"                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:                              - - GAATTCTTTT TTGCGTGTGG CAG           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:79:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "DNA"                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:                              - - ATCGATATTC CTTCCCCTTC C           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:80:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "DNA"                                    - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #difference                                       (B) LOCATION: replace(17, - #"")                                              (D) OTHER INFORMATION: - #/note= "The residue at this                              position - #can be repeated 18-21 times."                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:                              - - TCTAGAATTC ACGCGTN             - #                  - #                      - #   17                                                                 __________________________________________________________________________

What is claimed is:
 1. A method of producing a vaccine for treatment ofB-cell lymphoma comprising:a) providing:i) malignant B cells isolatedfrom a patient having a B-cell lymphoma; ii) an expression vector; iii)an amplification vector comprising a recombinant oligonucleotide havinga sequence encoding a first inhibitable enzyme operably linked to aheterologous promoter; and iv) a T lymphoid parent cell line; b)isolating nucleic acid from said malignant cells, said nucleic acidcomprising nucleotide sequences encoding at least one V_(H) region andat least one V_(L) region, said V_(H) and V_(L) regions derived fromimmunoglobulin molecules expressed by said malignant cells; c) insertingsaid nucleotide sequences encoding said V_(H) and V_(L) regions intosaid expression vector; d) introducing said expression vector and saidamplification vector into said parent cell line to generate one or moretransformed cells; e) growing said transformed cells in a first aqueoussolution containing an inhibitor capable of inhibiting said firstinhibitable enzyme wherein the concentration of said inhibitor presentin said first aqueous solution is sufficient to prevent growth of saidparent cell line; and f) identifying a transformed cell capable ofgrowth in said first aqueous solution, wherein said transformed cellcapable of growth expresses said V_(H) and V_(L) regions wherein V_(H)and V_(L) regions comprise a protein molecule useful as said vaccine. 2.The methods of claim 1, wherein transformed cell capable of growthcontains an amplified number of copies of said expression vector and anamplified number of copies of said amplification vector.
 3. The methodof claim 1, wherein nucleotide sequences encoding said V_(H) and V_(L)regions comprise at least two V_(H) and at least two V_(L) regions. 4.The method of claim 1, wherein said T lymphoid parent cell line is theBW5147.G.1.4 cell line.
 5. The method of claim 1, wherein said parentcell line contains an endogenous gene encoding a second inhibitableenzyme.
 6. The method of claim 5, wherein said second inhibitable enzymeis selected from the group consisting of dihydrofolate reductase,glutamine synthetase, adenosine deaminase, and asparagine synthetase. 7.The method of claim 1, wherein said concentration of inhibitor presentin said first aqueous solution is four to six-fold the concentrationrequired to prevent the growth of said parent cell line.
 8. The methodof claim 5, wherein said first and said second inhibitable enzyme arethe same.
 9. The method of claim 1, further comprising providing aselection vector encoding a selectable gene product which is introducedinto said parent cell line together with said expression vector and saidamplification vector.
 10. The method of claim 9, wherein said selectionvector encodes an active hypoxanthine guanine phosphoribosyltransferase.11. The method of claim 9, further comprising, following theintroduction of said vectors, the additional step of growing saidtransformed cell in a second aqueous solution which requires theexpression of said selectable gene product prior to growing saidtransformed cells in said first aqueous solution containing an inhibitorcapable of inhibiting said first inhibitable enzyme.
 12. The method ofclaim 11, wherein said second aqueous solution comprises hypoxanthineand azaserine.
 13. The method of claim 1, wherein said inhibitableenzyme encoded by said amplification vector is an active enzyme selectedfrom the group consisting of dihydrofolate reductase, glutaminesynthetase, adenosine deaminase, and asparagine synthetase.
 14. Themethod of claim 13, wherein said inhibitor is selected from the groupconsisting of methotrexate, 2'-deoxycoformycin, methionine sulphoximine,albizziin and β-aspartyl hydroxamate.
 15. The method of claim 1, whereinbetween approximately 20 and 30 micrograms of said amplification vectorand between approximately 400 and 500 micrograms of said expressionvector are introduced into said parent cell line.
 16. A method ofproducing a vaccine for treatment of B cell lymphoma, comprising:a)providing:i) malignant B cells isolated from a patient having a B-celllymphoma; ii) an expression vector; iii) an amplification vectorcomprising a first recombinant oligonucleotide having a sequenceencoding a first inhibitable enzyme operably linked to a heterologouspromoter; iv) a selection vector comprising a second recombinantoligonucleotide having a sequence which encodes a selectable geneproduct; and v) a T lymphoid parent cell line; b) isolating nucleic acidfrom said malignant cells, said nucleic acid comprising nucleotidesequences encoding at least one V_(H) region and at least one V_(L)region, said V_(H) and V_(L) regions derived from immunoglobulinmolecules expressed by said malignant cells; c) inserting saidnucleotide sequences encoding said V_(H) and V_(L) regions into saidexpression vector; d) introducing said expression vector, saidamplification vector and said selection vector into said parent cellline to generate transformed cells; e) introducing said transformedcells into a first aqueous solution, said first aqueous solutionrequiring the expression of said selectable gene product for growth ofsaid transformed cells; f) identifying at least one transformed cellcapable of growth in said first aqueous solution; g) introducing saidtransformed cell capable of growth in said first aqueous medium into asecond aqueous solution, said second aqueous solution comprising aninhibitor capable of inhibiting said first inhibitable enzyme, whereinthe concentration of said inhibitor present in said second aqueoussolution is sufficient to prevent growth of said parent cell line; andh) identifying at least one transformed cell capable of growth in saidsecond aqueous solution, wherein said transformed cell capable of growthexpresses said V_(H) and V_(L) regions wherein said V_(H) and V_(L)regions comprise a protein molecule.
 17. The method of claim 16, whereinsaid parent cell line contains an endogenous gene encoding a secondinhibitable enzyme.
 18. The method of claim 17, wherein said secondinhibitable enzyme is selected from the group consisting ofdihydrofolate reductase, glutamine synthetase, adenosine deaminase andasparagine synthetase.
 19. The method of claim 17, wherein said firstand said second inhibitable enzyme are the same.
 20. The method of claim16, wherein between approximately 10 and 15 micrograms of said selectionvector, between approximately 20 and 30 micrograms of said amplificationvector and between approximately 400 and 500 micrograms of saidexpression vector are introduced into said parent cell line.
 21. Themethod of claim 16, wherein said concentration of inhibitor present insaid second aqueous solution is four-fold to six-fold the concentrationrequired to prevent the growth of said parent cell line.
 22. The methodof claim 21 further comprising the steps of:i) introducing saidtransformed cell capable of growth in said second aqueous solution intoa third aqueous solution, said third aqueous solution comprising saidinhibitor capable of inhibiting said first inhibitable enzyme andwherein the concentration of said inhibitor present in said thirdaqueous solution is sixteen-fold to thirty-six-fold the concentration ofsaid inhibitor required to prevent the growth of said parent cell line;and j) identifying at least one transformed cell capable of growth insaid third aqueous solution.
 23. The method of claim 16, wherein saidselection vector encodes an active enzyme selected from the groupcomprising hypoxanthine guanine phosphoribosyltransferase, hygromycin Gphosphotransferase, xanthine-guanine phosphoribosyltransferase andaminoglycoside 3' phosphotransferase.
 24. The method of claim 16,wherein said T lymphoid cell line is the BW5147.G.1.4 cell line.
 25. Themethod of claim 16, wherein said inhibitable enzyme encoded by saidamplification vector is an active enzyme selected from the groupconsisting of dihydrofolate reductase, glutamine synthetase, adenosinedeaminase, asparagine synthetase.
 26. The method of claim 25, whereinsaid inhibitor is selected from the group consisting of methotrexate,2'-deoxycoformycin, methionine sulphoximine, albizziin and β-aspartylhydroxamate.
 27. The method of claim 16, wherein said expression,amplification and selection vectors are linearized prior to introductioninto said parent cell line.
 28. A method of producing a vaccine fortreatment of B cell lymphoma, comprising:a) providing:i) malignant Bcells isolated from a patient having a B-cell lymphoma; ii) anexpression vector; iii) an amplification vector comprising a firstrecombinant oligonucleotide having a sequence encoding a firstinhibitable enzyme operably linked to a heterologous promoter; iv) aselection vector comprising a second recombinant oligonucleotide havinga sequence which encodes a selectable gene product; and v) a T lymphoidparent cell line; b) isolating nucleic acid from said malignant cells,said nucleic acid comprising nucleotide sequences encoding at least oneV_(H) region and at least one V_(L) region, said V_(H) and V_(L) regionsderived from immunoglobulin molecules expressed by said malignant cells;c) inserting said nucleotide sequences encoding said V_(H) and V_(L)regions into said expression vector; d) introducing said expressionvector, said amplification vector and said selection vector into saidparent cell line to generate transformed cells; e) introducing saidtransformed cells into a first aqueous solution, said first aqueoussolution requiring the expression of said selectable gene product forgrowth of said transformed cells; f) identifying at least one individualclone of transformed cells capable of growth in said first aqueoussolution; g) introducing said individual clone capable of growth in saidfirst aqueous solution into a second aqueous solution, said secondaqueous solution comprising an inhibitor capable of inhibiting saidfirst inhibitable enzyme, wherein the concentration of said inhibitorpresent in said first aqueous solution is sufficient to prevent growthof said parent cell line; and h) identifying at least one individualclone capable of growth in said second aqueous solution, wherein saidclone capable of growth expresses said V_(H) and V_(L) regions whereinsaid V_(H) and V_(L) regions comprise a protein molecule.
 29. The methodof claim 28, wherein said parent cell line contains an endogenous geneencoding a second inhibitable enzyme.
 30. The method of claim 29,wherein said second inhibitable enzyme is selected from the groupconsisting of dihydrofolate reductase, glutamine synthetase, adenosinedeaminase and asparagine synthetase.
 31. The method of claim 29, whereinsaid first and said second inhibitable enzyme are the same.
 32. Themethod of claim 28, wherein between approximately 10 and 15 microgramsof said selection vector, between approximately 20 and 30 micrograms ofsaid amplification vector and between approximately 400 and 500micrograms of said expression vector are introduced into said parentcell line.
 33. The method of claim 28, wherein said concentration ofinhibitor present in said second aqueous solution is four-fold tosix-fold the concentration required to prevent the growth of said parentcell line.
 34. The method of claim 33 further comprising the steps of:i)introducing said transformed cell capable of growth in said secondaqueous solution into a third aqueous solution, said third aqueoussolution comprising said inhibitor capable of inhibiting said firstinhibitable enzyme and wherein the concentration of said inhibitorpresent in said third aqueous solution is sixteen-fold tothirty-six-fold the concentration of said inhibitor required to preventthe growth of said parent cell line; and j) identifying at least onetransformed cell capable of growth in said third aqueous solution. 35.The method of claim 28, wherein said selection vector encodes an activeenzyme selected from the group comprising hypoxanthine guaninephosphoribosyltransferase, hygromycin G phosphotransferase,xanthine-guanine phosphoribosyltransferase and aminoglycoside 3'phosphotransferase.
 36. The method of claim 28, wherein said T lymphoidparent cell line is the BW5147.G.1.4 cell line.
 37. The method of claim28, wherein said inhibitable enzyme encoded by said amplification vectoris an active enzyme selected from the group consisting of dihydrofolatereductase, glutamine synthetase, adenosine deaminase and asparaginesynthetase.
 38. The method of claim 27, wherein said inhibitor isselected from the group consisting of methotrexate, 2'-deoxycoformycin,methionine sulphoximine, albizziin and β-aspartyl hydroxamate.
 39. Themethod of claim 28, wherein said expression, amplification and selectionvectors are linearized prior to introduction into said parent cell line.40. A method of amplifying the number of copies of a vector,comprising:a) providing:i) a vector comprising a first recombinantoligonucleotide comprising nucleotide sequences encoding at least oneV_(H) region and at least one V_(L) region, said V_(H) and V_(L) regionsderived from immunoglobulin molecules expressed by malignant B cellsisolated from a patient having a B-cell lymphoma and a secondrecombinant oligonucleotide having a sequence encoding a firstinhibitable enzyme operably linked to a heterologous promoter; and ii) aT lymphoid parent cell line; b) introducing said vector into said Tlymphoid parent cell line to generate transformed cells; c) introducingsaid transformed cells into a first aqueous solution, said first aqueoussolution comprising an inhibitor capable of inhibiting said firstinhibitable enzyme, wherein the concentration of said inhibitor presentin said first aqueous solution is sufficient to prevent the growth ofsaid parent cell line; and d) identifying a transformed cell capable ofgrowth in said first aqueous solution, wherein said transformed cellcapable of growth contains an amplified number of copies of said vector.41. The method of claim 40, wherein said parent cell line contains anendogenous gene encoding a second inhibitable enzyme.
 42. The method ofclaim 41, wherein said second inhibitable enzyme is selected from thegroup consisting of dihydrofolate reductase, glutamine synthetase,adenosine deaminase and asparagine synthetase.
 43. The method of claim41, wherein said first and said second inhibitable enzyme are the same.44. The method of claim 40, wherein said concentration of inhibitorpresent in said second aqueous solution is four-fold to six-fold theconcentration required to prevent the growth of said parent cell line.45. The method of claim 44 further comprising the steps of:e)introducing said transformed cell capable of growth in said firstaqueous solution into a second aqueous solution, said second aqueoussolution comprising said inhibitor capable of inhibiting said firstinhibitable enzyme and wherein the concentration of said inhibitorpresent in said second aqueous solution is sixteen-fold tothirty-six-fold the concentration of said inhibitor required to preventthe growth of said parent cell line; and f) identifying at least onetransformed cell capable of growth in said second aqueous solution. 46.The method of claim 40 further comprising providing a selection vectorencoding a selectable gene product which is introduced into said parentcell line together with said vector comprising said first and secondrecombinant oligonucleotides.
 47. The method of claim 40, wherein saidvector is linearized prior to introduction into said parent cell line.